Transgenic fodder plants with an increased leaf starch content

ABSTRACT

There is described a method for generating transgenic fodder plants which are genetically modified, where the genetic modification leads to a reduced activity of an R1 protein in comparison to corresponding wild-type plants which have not been genetically modified. Such transgenic fodder plants are distinguished by their substantially increased leaf starch content in comparison with wild-type plants.

[0001] The present invention relates to a method for generating transgenic fodder plant cells or fodder plants which are genetically modified, the genetic modification in the cells or plants bringing about the reduced activity of an R1 protein in comparison to corresponding wild-type plant cells or plants which have not been genetically modified. Plants generated by this method synthesize a modified leaf starch, the starch content in the leaves of the plants being up to 1 000% higher than that of wild-type plants which have not been genetically modified. This starch is furthermore preferably characterized in that it has a reduced phosphate content. Furthermore, the present invention relates to transgenic fodder plant cells and fodder plants with a reduced activity of an R1 protein in comparison with wild-type plants or plant cells.

[0002] Fodder plants are mainly understood as meaning the clover-like fodder plants (=fodder legumes) and the fodder grasses. Both differ from most important crop plants by the fact that the crop product of interest is not predominantly reproductive organs such as seeds, fruits or tubers, but the entire aerial plant biomass. The fodder legumes include the clover species (such as, for example, the forms of Trifolium, Medicago and Lotus), the vetch species (such as Vicia and Coronilla), furthermore sainfoin (Esparsette) and serradella (Ornithopus). The fodder grasses include the various types of darnel, meadow-grass, cattail, Avena, cocksfoot grass and bentgrass, and the fescue species, inter alia.

[0003] The demands made of feed plants are an increased feed value. In addition to the consumption capacity, this complex term also encompasses the aspect of digestibility and the content of valuable constituents.

[0004] Only animals are capable of converting fodder plants properly. The farmer expects that an improved feed quality of the green fodder, which is provided in fresh form or as silage, will lead to better conversion of the material fed and that the energy requirements in livestock feeding will be met more specifically. However, the differences between ruminants and nonruminants must be taken into consideration. In cattle, the supplementation with concentrates only meets part of the energy requirements since the specific digestion processes in ruminants require a minimum of structured roughage. Fodder plants should therefore have a high utilizable energy content and ensure very good consumption. High energy requirement is in proportion to digestibility. Consumption depends on the rate of passage through the gut. Fodder plants, especially fodder legumes such as Trifolium repens and Lolium perenne, are very high in protein, but only contain a small amount of nonsoluble carbohydrates (NSC). As a consequence, most of the ammonium produced cannot be metabolized, but, in order to avoid poisoning, is transported to the liver, converted into uric acid and then excreted. This leads to nitrogen loss and simultaneously large amounts of excreted ammonia.

[0005] Livestock producers expect that an increased leaf starch content will lead to an improved carbon/nitrogen ratio (C/N) and, as a consequence, better digestibility and energy uptake and, eventually, higher production of meat/milk in combination with less excreted ammonia.

[0006] Ritte et al. (Plant J. 21 (4), (2000), 387-391) were able to demonstrate that, in potato plants, the Solanum tuberosum R1 protein binds reversibly to starch granules, the strength of binding to the starch granules depending on the metabolic status of the plant. In potato plants, the protein in its starch-granule-bound form occurs predominantly in leaves kept in the dark. After exposure of the leaves to light, in contrast, the protein occurs predominantly in the soluble form and is not bound to the starch granule.

[0007] Studies on transgenic potatoes have revealed that a reduced expression of the R1 gene leads to an increased starch content in the leaves of potato plants. This entails an increase in dry matter by 50-90%. Similar results were obtained in studies on leaves of transgenic tobacco plants in which the R1 gene was suppressed by antisense suppression (Lorberth et al., Nature Biotechnology 16 (1998), 473-477). However, no increased starch content in fodder plants have been shown as yet; in addition, the relevant gene sequences were not available.

[0008] The present invention is based on the object of providing a method by means of which transgenic fodder plants which are adapted better to the requirements of agriculture can be generated, in particular in as far as they have a higher utilizable energy content.

[0009] This object is achieved by providing the use forms specified in the patent claims.

[0010] The present invention thus relates to a method for generating a transgenic fodder plant with an increased leaf starch content in comparison to corresponding wild-type plants, where

[0011] (a) a cell of a fodder plant is genetically modified by introducing a foreign nucleic acid molecule whose presence or expression leads to a reduced activity of an R1 protein which occurs endogenously in the plant;

[0012] (b) a plant is regenerated from the cell generated in step (a); and

[0013] (c) if appropriate, further plants are generated starting from the plant generated in step (b).

[0014] In the present context, the term “transgenic” means that the plants generated by the method according to the invention differ, owing to a genetic modification, in particular the introduction of a foreign nucleic acid molecule, with regard to their genetic information from corresponding plant cells which have not been genetically modified and can be distinguished from them. In particular, the term means that the cells of these plants contain a foreign nucleic acid molecule which does not occur naturally in corresponding wild-type plants which have not been genetically modified or which occurs at a locus in the genome of the cells at which it does not occur naturally. In this context, the term “wild-type plants” means a plant of the same species which, however, contains no corresponding genetic modification, in particular no genetic modification in connection with the R1 gene. In this context, “foreign” nucleic acid molecule means that the nucleic acid molecule is heterologous with regard to the species to which the plants into whose cells the nucleic acid molecule is introduced belong, or that, if the nucleic acid molecule is homologous to this plant species, it occurs in a genetic context in which it does not occur naturally in the plant cells. This means that it occurs at a different locus in the genome of the plant cell and/or is linked to sequences to which it is not linked naturally in the plant cells. Whether a plant or plant cell is transgenic can be verified by methods with which the skilled worker is familiar, for example Southern blot analysis.

[0015] For the purposes of the present invention, the term “fodder plants” refers to plants which are grown for the purpose of being utilized as animal feed.

[0016] The “fodder plants” may take the form of monocotyledonous or else dicotyledonous fodder plants. In particular, “fodder plants” is understood as meaning what are known as fodder legumes. These are plants which belong to the superorder of the Fabanae, in particular the Fabales (=Leguminosae). Preferred in this context are plants of the Fabaceae family. Especially preferred are clover species, for example plants of the genus Trifolium, such as, for example, T. repens, T. pratense, T. hybridum or T. incarnatum. Others which are especially preferred are Lucerne (Medicago sativa), Lotus types, for example Lotus corniculatus; sainfoin (Onobrychis viciifolia), Serradella (Ornithopus sativus), lupin, for example Lupinus angustifolius or Lupinus luteus, and vetch species, such as, for example, Vicia species (for example Vicia faba) or Coronilla species.

[0017] Fodder plants are furthermore understood as meaning fodder grasses. These are plants of the order Poales, in particular those of the Poaceae family. Preferred in this context are plants of the genus Lolium (for example Lolium perenne), Poa (for example P. pratensis, P. palustris, P. longifolia), Phleum (for example P. nodusum, P. pratense), Dactylis (for example D. glomerata), Agrostis (for example A. tennuis, A. stolonifera), Festuca (for example F. pratensis, F. rubra), Bromus (for example Bromus molli) and Avena species.

[0018] The term “increased leaf starch content” refers to the fact that the amount of starch formed in the leaves markedly exceeds the amount formed in the leaves of corresponding wild-type plants. The leaf starch content of the fodder plants generated by the method according to the invention is increased by at least 10-50%, preferably by at least 50-100%, in particular by 100-500% and very particularly preferably by at least 500-1 000% in comparison with the leaf starch content of corresponding wild-type plants.

[0019] The starch content in the leaf is preferably measured in nmol/g dry matter. Methods for determining the leaf starch content are known to the skilled worker and described for example in the examples which follow.

[0020] For the purposes of the present invention, the term “genetically modified” refers to the fact that the genetic information of the plant cell is modified in comparison with corresponding cells of a wild-type plant by the introduction of a foreign nucleic acid molecule and that the presence and/or the expression of the foreign nucleic acid molecule results in an altered phenotype of the plant regenerated from this plant cell. In this context, altered phenotype preferably means a measurable difference of one or more functions of the cell and/or the plant. By way of example, in genetically modified plant cells according to the invention, the activity of an R1 protein which occurs endogenously in the plant cell is reduced in comparison with corresponding plant cells of wild-type plants which have not been genetically modified.

[0021] For the purposes of the present invention, the term “reduced activity” refers to a reduced expression of endogenous genes which encode R1 proteins, and/or to a reduced amount of R1 protein in the cells and/or a reduced biological activity of the R1 proteins in the cells.

[0022] In this context, “reduced expression” refers to the fact that plants generated by the method according to the invention, or the cells of these plants, contain fewer transcripts which encode an R1 protein than corresponding wild-type plant cells. “Reduced expression” can be determined for example by measuring the amount of transcripts encoding R1 proteins, for example by Northern blot analysis or RT-PCR. Reduction in this context preferably means a reduced amount of transcripts by at least 50%, in particular by at least 70%, preferably by at least 85% and especially preferably by at least 95% in comparison with corresponding cells which have not been genetically modified. In a very especially preferred embodiment, the reduction amounts to 100%, i.e. the expression of R1 genes in the plants (plant cells) is completely repressed, and no R1 protein whatsoever is synthesized in the cells.

[0023] “Reduced amount” of R1 protein means that the content of R1 protein in the plants or in the cells of the plants which are generated by the method according to the invention is less than in corresponding wild-type plants or wild-type plant cells. Methods for determining the R1 protein content are known to the skilled worker.

[0024] Thus, the reduced amount of R1 proteins can be determined for example by Western blot analysis. In this context, reduction means a reduced amount of R1 protein by at least 50%, in particular by at least 70%, preferably by at least 85%, especially preferably by at least 95% and very especially preferably by 100% in comparison to corresponding wild-type plants or cells which have not been genetically modified.

[0025] For the purposes of the present invention, the term “R1 gene” is understood as meaning a nucleic acid sequence (for example RNA or DNA, such as cDNA or genomic DNA) which encodes a “R1 protein”. For the purposes of the present invention, the term “R1 protein” is understood as meaning proteins which have been described for example in Lorberth et al. (Nature Biotech. 16 (1998), 473-477) and in the international patent applications WO 98/27212, WO 00/77229 and WO 00/28052 and which have specific traits. Important traits of R1 proteins are

[0026] (i) their localization in the plastids (such as chloroplasts, amyloplasts) of plant cells;

[0027] (ii) their property of occurring, in the plastids, partly in free form and partly bound to starch granules;

[0028] (iii) their ability of influencing the degree of starch phosphorylation in plants, inasmuch as an increased activity of the R1 protein in plants leads to an increased phosphate content of the starch synthesized in the plants, and a reduced activity of the R1 protein in plants leads to a reduced phosphate content of the starch synthesized in the plants. In this context, the phosphate content relates to the C-6-phosphate content. It is preferably indicated as nmol/mg dry leaf starch. It can be determined as described in the examples section which follows; and

[0029] (iv) their ability of, when expressed in E. coli cells, leading to phosphorylation of the bacterial glycogen. This ability can be assayed for example as described in WO 98/27212.

[0030] For the purposes of the present invention, an R1 protein is preferably understood as meaning a protein which has the abovementioned characteristics and which is encoded by a nucleic acid molecule encompassing a nucleotide sequence which hybridizes with the coding region of SEQ ID NO: 7 or its complementary strand. In this context, the term “hybridization” refers to hybridization under conventional hybridization conditions, preferably under stringent conditions as are described, for example, in Sambrook et al. (1989, Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Such hybridizing nucleic acid molecules can be isolated for example from genomic libraries or from cDNA libraries of plants.

[0031] Such nucleic acid molecules can be identified and isolated using the nucleic acid molecules shown in SEQ ID NO: 7 or parts of these molecules or the reverse complements of these molecules, for example by means of hybridization by standard methods (see, for example, Sambrook and Russel, Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press (2001), Cold Spring Harbor, N.Y.).

[0032] Examples of nucleic acid molecules which can be used as hybridization probe are those with exactly or essentially the sequence shown under SEQ ID NO: 7 or part of this sequence. The DNA fragments used as hybridization probe may also take the form of synthetic DNA fragments which have been generated with the aid of the customary DNA synthesis techniques and whose sequence agrees essentially with the nucleic acid sequence shown in SEQ ID NO: 7.

[0033] “Hybridization” preferably means that at least 60%, preferably at least 80%, especially preferably at least 90% and very especially preferably at least 95% homology, i.e. sequence identity, exists between the molecules in question.

[0034] The degree of homology is determined by comparing the nucleotide sequence in question with the coding region shown in SEQ ID NO: 7. If the sequences to be compared differ in length, the degree of homology preferably relates to the percentage of nucleotides in the shorter sequence which are identical to nucleotides in the longer sequence. The homology can be determined by customary methods, in particular using computer programs such as, for example, the DNASTAR program in combination with Clustal W analysis. This program is obtainable from DNASTAR, Inc. 1228 South Park Street, Madison, Wis. 53715 or from DNASTAR, Ltd., Abacus House, West Ealing, London W13 OAS UK (support @dnastar.com), and is accessible via the EMBL server.

[0035] If the analytical method Clustal is used for determining homology, in particular for determining whether a sequence is identical, for example 80% identical, to a reference sequence, the settings are preferably as follows: Matrix: blosum 30; Open gap penalty: 10.0; Extend gap penalty: 5.0; Delay divergent: 40; Gap separation distance 8.

[0036] Generation of the plant cells with reduced R1 activity in accordance with the method according to the invention can be achieved by various methods with which the skilled worker is familiar, for example by those which lead to inhibition of the expression of endogenous genes which encode an R1 protein. These include, for example, the expression of a corresponding antisense RNA, the provision of molecules or vectors which confer a cosuppression effect, the expression of a suitably constructed ribozyme which specifically cleaves transcripts encoding an R1 protein, or what is known as “in-vivo mutagenesis”.

[0037] Furthermore, reduced R1 activity in the plant cells can also be brought about by the simultaneous expression of sense and antisense RNA molecules of the target gene in question which is to be repressed, preferably of the R1 gene. Moreover, it is known that the formation, in planta, of RNA duplex molecules of promoter sequences in trans can lead to methylation and transcriptional inactivation of homologous copies of this promoter (Mette et al., EMBO J. 19 (2000), 5194-5201).

[0038] Furthermore, the use of introns, i.e. of noncoding regions of genes which encode R1 proteins, is also feasible for achieving an antisense or a cosuppression effect. The use of intron sequences for inhibiting the gene expression of genes which encode proteins of starch biosynthesis has been described for example in international patent applications WO 97/04112, WO 97/04113, WO 98/37213 and WO 98/37214.

[0039] In a preferred embodiment of the method according to the invention, the foreign nucleic acid molecule is selected from the group consisting of

[0040] (a) DNA molecules which encode at least one antisense RNA which brings about reduced expression of endogenous genes which encode R1 proteins;

[0041] (b) DNA molecules which, via a cosuppression effect, lead to reduced expression of endogenous genes which encode R1 proteins;

[0042] (c) DNA molecules which encode at least one ribozyme which specifically cleaves transcripts of endogenous genes which encode R1 proteins;

[0043] (d) nucleic acid molecules which, in the event of in vivo mutagenesis, lead to a mutation or an insertion of a heterologous sequence in endogenous genes which encode R1 proteins, the mutation or insertion leading to reduced expression of genes encoding R1 proteins, or to a reduced synthesis of R1 proteins; and

[0044] (e) DNA molecules which simultaneously encode at least one antisense RNA and at least one sense RNA, where the antisense RNA and the sense RNA form an RNA duplex which brings about a reduced expression of endogenous genes which encode R1 proteins.

[0045] The skilled worker knows how to achieve an antisense effect or a cosuppression effect. The method of antisense inhibition is described, for example, in Krol et al. (Nature 333 (1988), 866-869), Krol et al. (Gene 72 (1988), 45-50), Mol et al. (FEBS Letters 268 (2), (1990), 427-430), Smith et al. (Plant Mol. Biol. 14 (1990), 369-379) and Sheehy et al. (Proc. Natl. Acad. Sci. USA 85 (1988), 8805-8809). The method of cosuppression inhibition has been described, for example, in Jorgensen (Trends Biotechnol. 8 (1990), 340-344), de Carvalho Niebel et al. (Curr. Top. Microbiol. Immunol. 197 (1995), 91-103), Flavell et al. (Curr. Top. Microbiol. Immunol. 197 (1995), 43-56), Palauqui and Vaucheret (Plant. Mol. Biol. 29 (1995), 149-159), Vaucheret et al. (Mol. Gen. Genet. 248 (1995), 311-317) and in de Borne et al. (Mol. Gen. Genet. 243 (1994), 613-621).

[0046] The expression of ribozymes for reducing the activity of certain enzymes in cells is likewise known to the skilled worker and described, for example, in EP-B1 0321 201. The expression of ribozymes in plant cells has been described, for example, in Feyter et al. (Mol. Gen. Genet. 250 (1996), 329-338).

[0047] Furthermore, the reduced R1 activity in the plant cells can also be achieved, as mentioned above, by what is known as “in-vivo mutagenesis”, where a chimeric RNA-DNA oligonucleotide (“chimeroplast”) is introduced into cells by cell transformation (Beetham, Kipp et al., Poster Session at the “5^(th) International Congress of Plant Molecular Biology”, Sep. 21-27, 1997, Singapore; Dixon and Arntzen, Meeting report on “Metabolic Engineering in Transgenic Plants”, Keystone Symposia, Copper Mountain, Colo., USA, TIBTECH 15 (1997), 441-447; international patent application WO 95/15972; Kren et al., Hepatology 25 (1997), 1462-1468; Cole-Strauss et al., Science 273 (1996), 1386-1389).

[0048] Part of the DNA component of the RNA-DNA oligonucleotide is homologous to a nucleic acid sequence of an endogenous R1 gene, but contains a mutation in comparison with the nucleic acid sequence of an endogenous R1 gene or contains a heterologous region which is flanked by the homologous region. Owing to this pairing of the homologous regions of the RNA-DNA oligonucleotide and of the endogenous nucleic acid molecule, followed by homologous recombination, the heterologous region or the mutation obtained in the DNA component of the RNA-DNA oligonucleotide can be transferred into the genome of a plant cell. The mutation is chosen in such a way that it, upon expression of the corresponding sequence, leads to a reduced activity of an R1 protein, i.e. preferably to a reduced expression of the gene, to a reduced synthesis of an R1 protein or to a reduced biological activity of an R1 protein, for example owing to the expression of inactive proteins, in particular of dominant-negative mutants.

[0049] Furthermore, the reduced R1 activity in the plant cells may also be brought about by the simultaneous expression of sense and antisense RNA molecules of the target gene to be repressed, i.e. of the R1 gene. This can be achieved for example by using chimeric constructs which contain inverted repeats of the target gene to be repressed, or of parts of the target gene. In this context, the chimeric constructs encode sense and antisense RNA molecules of the target gene. Sense and antisense RNA are synthesized simultaneously in planta as an RNA molecule, it being possible for sense and antisense RNA to be separated from each other by a spacer, forming an RNA duplex. It has been demonstrated that the introduction of inverted-repeat DNA constructs into the genome of plants is a highly effective method of repressing the genes which correspond with the inverted-repeat DNA constructs (Waterhouse et al., Proc. Natl. Acad. Sci. USA 95 (1998), 13959-13964; Wang and Waterhouse, Plant Mol. Biol. 43 (2000), 67-82; Singh et al., Biochemical Society Transactions 28, part 6 (2000), 925-927; Liu et al., Biochemical Society Transactions 28, part 6 (2000), 927-929); Smith et al., Nature 407 (2000), 319-320; international patent application WO 99/53050). Sense and antisense sequences of the target gene(s) may also be expressed separately of one another by means of identical or different promoters (Nap et al., 6^(th) International Congress of Plant Molecular Biology, Quebec, Jun. 18-24, 2000; Poster S7-27, paper Session S7).

[0050] Thus, reduced R1 activity in the plant cells can also be achieved by generating RNA duplexes of R1 genes. For this purpose, it is preferred to introduce, into the genome of plants, inverted repeats of DNA molecules of R1 genes or R1 cDNAs, the DNA molecules to be transcribed (R1 gene or R1 cDNA or fragments thereof) being under the control of a promoter which governs the expression of said DNA molecules.

[0051] Moreover, it is known that the formation of RNA duplexes of promoter DNA molecules in plants in trans can lead to methylation and a transcriptional inactivation of homologous copies of these promoters, which are hereinbelow referred to as target promoters (Mette et al., EMBO J. 19 (2000), 5194-5201). Thus, it is possible, via the inactivation of the target promoter, to reduce the gene expression of a particular target gene (for example of the R1 gene) which is naturally under the control of this target promoter. This means that the DNA molecules which comprise the target promoters of the genes to be repressed (target genes) are now not used as control elements for expressing genes or cDNAs, but as transcribable DNA molecules themselves, which is in contrast to the original function of promoters in plants.

[0052] Constructs which are preferably used for generating the target promoter RNA duplexes in planta, where they may exist in the form of RNA hairpin molecules, are those comprising inverted repeats of the target promoter DNA molecules, the target promoter DNA molecules being under the control of a promoter which governs the gene expression of said target promoter DNA molecules. These constructs are subsequently introduced into the genome of plants. In planta, the expression of the inverted repeats of said target promoter DNA molecules leads to the formation of target promoter RNA duplexes (Mette et al., EMBO J. 19 (2000), 5194-5201), by which the target promoter can be inactivated.

[0053] The promoter regions of the R1 genes from the plant species in question can be isolated and characterized by screening suitable genomic DNA libraries. Known cDNA or genomic fragments of the R1 genes can be used as homologous probes in this context. The generation and screening of genomic DNA libraries is known to the skilled worker and described in Sambrook and Russell (Molecular Cloning, 3rd edition (2001), Cold Spring Harbour Laboratory Press, NY).

[0054] Furthermore, the skilled worker knows that a reduced activity of one or more R1 proteins can be achieved by the expression of nonfunctional derivatives, in particular trans-dominant mutants of such proteins, and/or by the expression of antagonists/inhibitors of those proteins. Antagonists/inhibitors of those proteins encompass for example antibodies, antibody fragments or molecules with similar binding properties. For example, a cytoplasmic scFv antibody was employed for modulating the activity of the phytochrome A protein in genetically modified tobacco plants (Owen, Bio/Technology 10 (1992), 790-794; Review: Franken et al., Current Opinion in Biotechnology 8 (1997), 411-416; Whitelam, Trends in Plant Science 1 (8), (1996), 268-272).

[0055] In an especially preferred embodiment of the method according to the invention, the gene which occurs endogenously in the fodder plant and which encodes an R1 protein is selected from the groups consisting of:

[0056] (a1) nucleic acid molecules which encompass the coding region of the nucleotide sequence shown under SEQ ID NO: 1 or the coding region of the insertion in plasmid DSM 14707;

[0057] (b1) nucleic acid molecules which encode a protein encompassing the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence encoded by the insertion of plasmid DSM 14707;

[0058] (c1) nucleic acid molecules whose sequence has at least 90% homology with the nucleic acid molecules mentioned under (a1) or (b1);

[0059] (d1) nucleic acid molecules whose sequence is degenerate in comparison with the sequences of the nucleic acid molecules mentioned under (a1), (b1) or (c1) owing to the genetic code; and

[0060] (e1) parts or fragments of the nucleic acid molecules mentioned under a1) to d1) selected from the group consisting of fragments with a length of at least 25, 50, 100 bp, preferably with a length of at least 250 bp, especially preferably with a length of at least 500 bp.

[0061] In one such embodiment, the plant generated in the method according to the invention is preferably a plant of the genus Trifolium, especially preferably of the species Trifolium repens. Furthermore, the foreign nucleic acid molecule preferably employed in such an embodiment is a nucleic acid molecule which is a nucleic acid molecule as defined above under (a1) to (d1), part of such a molecule which is long enough in order to achieve the desired effect, or a nucleic acid molecule which is complementary to such a nucleic acid molecule or part thereof.

[0062] The sequence shown under SEQ ID NO: 1 originates from Trifolium repens and constitutes a partial cDNA sequence which encodes an R1 protein. The coding region shown has 75% homology with the coding region shown in SEQ ID NO: 7, which encodes the potato R1 protein.

[0063] In another preferred embodiment, the gene which occurs endogenously in the fodder plant and which encodes an R1 protein is a gene selected from the group consisting of:

[0064] (a2) nucleic acid molecules which encompass the coding region of the nucleotide sequence shown under SEQ ID NO: 3 or the coding region of the insertion in plasmid DSM 14633;

[0065] (b2) nucleic acid molecules which encode a protein encompassing the amino acid sequence shown in SEQ ID NO: 4 or the amino acid sequence encoded by the insertion of plasmid DSM 14633;

[0066] (c2) nucleic acid molecules whose sequence has at least 90% homology with the nucleic acid molecules mentioned under (a2) or (b2);

[0067] (d2) nucleic acid molecules whose sequence is degenerate in comparison with the sequences of the nucleic acid molecules mentioned under (a2), (b2) or (c2) owing to the genetic code; and

[0068] (e2) parts or fragments of the nucleic acid molecules mentioned under a2) to d2) selected from the group consisting of fragments with a length of at least 25, 50, 100 bp, preferably with a length of at least 250 bp, especially preferably with a length of at least 500 bp.

[0069] In one such embodiment, the plant generated in the method according to the invention is preferably a plant of the genus Medicago, especially preferably of the species Medicago sativa. Furthermore, the foreign nucleic acid molecule preferably employed in such an embodiment is a nucleic acid molecule which is a nucleic acid molecule as defined above under (a2) to (d2), part of such a molecule which is long enough in order to achieve the desired effect, or a nucleic acid molecule which is complementary to such a nucleic acid molecule or part thereof.

[0070] The sequence shown under SEQ ID NO: 3 originates from Medicago sativa and constitutes a partial cDNA sequence which encodes an R1 protein. The coding region shown has 76% homology with the coding region shown in SEQ ID NO: 7, which encodes the potato R1 protein.

[0071] In another preferred embodiment of the method according to the invention, the gene which occurs endogenously in the food plant and which encodes an R1 protein is a gene selected from the group consisting of:

[0072] (a3) nucleic acid molecules which encompass the coding region of the nucleotide sequence shown under SEQ ID NO: 5 or the coding region of the insertion in plasmid DSM 14635;

[0073] (b3) nucleic acid molecules which encode a protein encompassing the amino acid sequence shown in SEQ ID NO: 6 or the amino acid sequence encoded by the insertion of plasmid DSM 14635;

[0074] (c3) nucleic acid molecules whose sequence has at least 90% homology with the nucleic acid molecules mentioned under (a3) or (b3);

[0075] (d3) nucleic acid molecules whose sequence is degenerate in comparison with the sequences of the nucleic acid molecules mentioned under (a3), (b3) or (c3) owing to the genetic code; and

[0076] (e3) parts or fragments of the nucleic acids mentioned under a3) to d3) selected from the group consisting of fragments with a length of at least 25, 50, 100 bp, preferably with a length of at least 250 bp, especially preferably with a length of at least 500 bp.

[0077] In one such embodiment, the fodder plant generated in the method according to the invention is preferably a fodder plant of the genus Lolium, especially preferably of the species Lolium perenne. Furthermore, the foreign nucleic acid molecule preferably employed in such an embodiment is a nucleic acid molecule which is a nucleic acid molecule as defined above under (a3) to (d3), part of such a molecule which is long enough in order to achieve the desired effect, or a nucleic acid molecule which is complementary to such a nucleic acid molecule or part thereof.

[0078] The sequence shown under SEQ ID NO: 5 originates from Lolium perenne and constitutes a partial cDNA sequence which encodes an R1 protein. The coding region shown has 75% homology with the coding region shown in SEQ ID NO: 7, which encodes the potato R1 protein.

[0079] The present invention also relates to the transgenic fodder plants generated by the method according to the invention, which have an increased leaf starch content in comparison with wild-type plants. The leaf starch content of the fodder plants according to the invention is increased by at least 10-50%, preferably by at least 50-100%, in particular by at least 100-500% and very particularly preferably by at least 500-1 000% in comparison with the corresponding wild-type plants.

[0080] In a preferred embodiment, the fodder plants according to the invention are furthermore characterized in that the leaf starch synthesized in them has a reduced phosphate content in comparison with leaf starch from corresponding wild-type plants. In this context, the term “reduced phosphate content” refers to the fact that the phosphate content is reduced by at least 25%, especially preferably by at least 50% and very especially preferably by at least 100% in comparison to the phosphate content of the leaf starch from corresponding wild-type plants. The phosphate content of the leaf starch can be determined by methods known to the skilled worker. It is preferably determined as described in the examples section which follows.

[0081] The present invention also relates to cells of a fodder plant according to the invention, the cells being transgenic and genetically modified by the introduction of a foreign nucleic acid molecule whose presence or expression leads to a reduced activity of an R1 protein which occurs endogenously in the fodder plant.

[0082] In connection with the transgenic fodder plants and plant cells according to the invention, what has already been said above in connection with the method according to the invention also applies to the preferred embodiments.

[0083] A multiplicity of techniques is available for introducing the foreign nucleic acid molecule into a plant host cell in accordance with step (a) of the method according to the invention. These techniques encompass the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, protoplast fusion, injection, the electroporation of DNA, the introduction of the DNA by means of the biolistic approach, and other possibilities.

[0084] The use of agrobacteria-mediated transformation of plant cells has been studied intensively and described sufficiently, for example in EP 120 516; Hoekema, in: The Binary Plant Vector System, Offsetdrukkerij Kanters B. V., Alblasserdam (1985), chapter V, 63-71; Fraley et al., Crit. Rev. Plant Sci. 4, 1-46 and in An et al., EMBO J. 4 (2), (1985), 277-284. As regards the transformation of potato, see, for example, Rocha-Sosa et al., EMBO J. 8 (1), (1989), (23-29).

[0085] The transformation of monocotyledonous plants by means of vectors based on Agrobacterium has also been described (Chan et al., Plant Mol. Biol. 22 (1993), 491-506; Hiei et al., Plant J. 6 (1994) 271-282; Deng et al., Science in China 33 (1), (1990), 28-34; Wilmink et al., Plant Cell Reports 11 (1992), 76-80; May et al., Bio/Technology 13 (1995), 486-492; Conner and Domisse, Int. J. Plant Sci. 153 (1992), 550-555; Ritchie et al., Transgenic Res. 2 (1993), 252-265). An alternative system for the transformation of monocotyledonous plants is the transformation by means of the biolistic approach (Wan and Lemaux, Plant Physiol. 104 (1994), 37-48; Vasil et al., Bio/Technology 11 (1993), 1553-1558; Ritala et al., Plant Mol. Biol. 24 (1994), 317-325; Spencer et al., Theor. Appl. Genet. 79 (1990), 625-631), protoplast transformation, the electroporation of partially permeabilized cells, or the introduction of DNA by means of glass fibres. In particular the transformation of maize is described repeatedly in the literature (cf., for example, WO 95/06128, EP 0 513 849, EP 0 465 875, EP 0 292 435; Fromm et al., Biotechnology 8 (1990), 833-839; Gordon-Kamm et al., Plant Cell 2 (1990), 603-618; Koziel et al., Biotechnology 11 (1993), 194-200; Mórocz et al., Theor. Appl. Genet. 80 (1990), 721-726).

[0086] The successful transformation of other cereal species has also been described, for example for barley (Wan and Lemaux, above; Ritala et al., above; Krens et al., Nature 296 (1982), 72-74) and for wheat (Nehra et al., Plant J. 5 (2), (1994), 285-297; Altpeter et al., Mol. Breeding 6 (2000), 519-528).

[0087] The genetically modified cell may be regenerated into a plant in accordance with step (b) of the method according to the invention by prior-art methods with which the skilled worker is familiar.

[0088] If the expression of the foreign nucleic acid molecule in the plant cells is required for achieving the desired effect, i.e. a reduced activity of the R1 protein, any promoter which is active in plant cells is suitable for this purpose. The promoter can be chosen in such a way that expression in the plants is constitutive or only takes place in a particular tissue, at a particular point in time of plant development or at a point in time determined by external factors. With regard to the plant, the promoter can be homologous or heterologous. Useful promoters are, for example, the cauliflower mosaic virus 35S RNA promoter, the maize ubiquitine promoter and the actin promoter for constitutive expression, or a promoter which ensures expression only in photosynthetically active tissues, for example the ST-LS1 promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO J. 8 (9), (1989), 2445-2451), the Ca/b promoter (see, for example, U.S. Pat. No. 5,656,496; U.S. Pat. No. 5,639,952; Bansal et al., Proc. Natl. Acad. Sci. USA 89 (1992), 3654-3658) and the Rubisco SSU promoter (see, for example, U.S. Pat. No. 5,034,322 or U.S. Pat. No. 4,962,028), and inducible promoters.

[0089] Furthermore, the foreign nucleic acid molecule may contain a termination sequence which serves for the correct termination of transcription and for the addition of a poly-A tail to the transcript, which is thought to have a function in stabilizing the transcripts. Such elements are described in the literature (cf., for example, Gielen et al., EMBO J. 8 (1), (1989), 23-29) and can be exchanged as desired.

[0090] The generation of further plants in accordance with step (c) of the method according to the invention can be carried out by any suitable method, for example by vegetative propagation (for example via cuttings, tubers or via callus culture and regeneration of intact plants) or by sexual propagation. Sexual propagation preferably takes place under controlled circumstances, i.e. selected plants which have specific characteristics are hybridized with each other and propagated. The present invention also relates to the plants which can be obtained by this type of propagation.

[0091] The present invention also encompasses propagation material of the fodder plants according to the invention, comprising the plant cells according to the invention. For the purposes of the present invention, the term “propagation material” encompasses those components of the plant which are suitable for generating progeny via the vegetative or generative route. Examples which are suitable for vegetative propagation are cuttings, callus cultures or rhizomes. Other propagation material encompasses, for example, fruit, seeds, seedlings, protoplasts, cell cultures and the like. The preferred propagation materials is seeds.

[0092] Moreover, the present invention also encompasses the use of nucleic acid molecules which encode an R1 protein, or of parts of these, for reducing the activity of an R1 protein in fodder plants, and to the use of such nucleic acid molecules for generating transgenic fodder plants with an increased leaf starch content in comparison with wild-type plants.

[0093] The present invention furthermore relates to nucleic acid molecules encoding an R1 protein from plants of the genus Trifolium, selected from the group consisting of

[0094] (a) nucleic acid molecules which encode a protein encompassing the amino acid sequence shown in SEQ ID NO: 2;

[0095] (b) nucleic acid molecules which encompass the nucleotide sequence, shown in SEQ ID NO: 1, of the coding region;

[0096] (c) nucleic acid molecules which encode a protein which encompasses the amino acid sequence encoded by the insertion of plasmid DSM 14707;

[0097] (d) nucleic acid molecules which encompass a region, of the insertion of plasmid DSM 14707, which encodes a Trifolium repens R1 protein;

[0098] (e) nucleic acid molecules whose sequence has at least 85%, by preference at least 90%, preferably at least 95%, especially preferably at least 97% and very especially preferably at least 99% identity with the sequence of a nucleic acid molecule of (a), (b), (c) and/or (d) and which nucleic acid molecules encode an R1 protein from a plant of the genus Trifolium; and

[0099] (f) nucleic acid molecules whose nucleotide sequence deviates from the sequence of a nucleic acid molecule of (e) owing to the degeneracy of the genetic code.

[0100] The nucleic acid sequence shown in SEQ ID NO: 1 is a partial cDNA sequence which encompasses part of the coding region for a Trifolium repens R1 protein. A plasmid comprising this cDNA sequence was deposited as DSM 14707. This sequence, or this molecule, now enables the skilled worker to isolate homologous sequences from other Trifolium varieties. This can be done for example with the aid of conventional methods, such as screening cDNA libraries or genomic libraries with suitable hybridization probes. In this manner, it is possible, for example, to identify and isolate nucleic acid molecules which hybridize, and have at least 85% identity, with the sequence shown under SEQ ID NO: 1 and which encode a Trifolium R1 protein.

[0101] In principle, the nucleic acid molecules according to the invention can encode an R1 protein from any desired plant of the genus Trifolium; preferably, they encode a Trifolium repens R1 protein.

[0102] The present invention furthermore relates to nucleic acid molecules encoding an R1 protein from plants of the genus Medicago, selected from the group consisting of

[0103] (a) nucleic acid molecules which encode a protein encompassing the amino acid sequence shown in SEQ ID NO: 4;

[0104] (b) nucleic acid molecules which encompass the nucleotide sequence, shown in SEQ ID NO: 3, of the coding region;

[0105] (c) nucleic acid molecules which encode a protein which encompasses the amino acid sequence encoded by the insertion of plasmid DSM 14633;

[0106] (d) nucleic acid molecules which encompass a region, of the insertion of plasmid DSM 14633, which encodes a Medicago sativa R1 protein;

[0107] (e) nucleic acid molecules whose sequence has at least 80%, by preference at least 85%, preferably at least 90%, especially preferably at least 95% and very especially preferably at least 99% identity with the sequence of a nucleic acid molecule of (a), (b), (c) and/or (d) and which nucleic acid molecules encode an R1 protein from a plant of the genus Medicago; and

[0108] (f) nucleic acid molecules whose nucleotide sequence deviates from the sequence of a nucleic acid molecule of (e) owing to the degeneracy of the genetic code.

[0109] The nucleic acid sequence shown in SEQ ID NO: 3 is a partial cDNA sequence which encompasses part of the coding region for a Medicago sativa R1 protein. A plasmid comprising this cDNA sequence was deposited as DSM 14633. This sequence, or this molecule, now enables the skilled worker to isolate homologous sequences from other Medicago species or varieties. This can be done for example with the aid of conventional methods, such as screening cDNA libraries or genomic libraries with suitable hybridization probes. In this manner, it is possible, for example, to identify and isolate nucleic acid molecules which hybridize, and have at least 80% identity, with the sequence shown under SEQ ID NO: 3 and which encode a Medicago R1 protein.

[0110] In principle, the nucleic acid molecules according to the invention can encode an R1 protein from any desired plant of the genus Medicago; preferably, they encode a Medicago sativa R1 protein.

[0111] The present invention furthermore relates to nucleic acid molecules encoding an R1 protein from plants of the genus Lolium, selected from the group consisting of

[0112] (a) nucleic acid molecules which encode a protein encompassing the amino acid sequence shown in SEQ ID NO: 6;

[0113] (b) nucleic acid molecules which encompass the nucleotide sequence, shown in SEQ ID NO: 5, of the coding region;

[0114] (c) nucleic acid molecules which encode a protein which encompasses the amino acid sequence encoded by the insertion of plasmid DSM 14635;

[0115] (d) nucleic acid molecules which encompass a region, of the insertion of plasmid DSM 14635, which encodes a Lolium perenne R1 protein;

[0116] (e) nucleic acid molecules whose sequence has at least 90%, by preference at least 95%, preferably at least 97%, especially preferably at least 99% identity with the sequence of a nucleic acid molecule of (a), (b), (c) and/or (d) and which nucleic acid molecules encode an R1 protein from a plant of the genus Lolium; and

[0117] (f) nucleic acid molecules whose nucleotide sequence deviates from the sequence of a nucleic acid molecule of (e) owing to the degeneracy of the genetic code.

[0118] The nucleic acid sequence shown in SEQ ID NO: 5 is a partial cDNA sequence which encompasses part of the coding region for a Lolium perenne R1 protein. A plasmid comprising this DNA sequence was deposited as DSM 14635. This sequence, or this molecule, now enables the skilled worker to isolate homologous sequences from other Lolium species or varieties. This can be done for example with the aid of conventional methods, such as screening cDNA libraries or genomic libraries with suitable hybridization probes. In this manner, it is possible, for example, to identify and isolate nucleic acid molecules which hybridize, and have at least 90% identity, with the sequence shown under SEQ ID NO: 5 and which encode a Lolium R1 protein.

[0119] In principle, the nucleic acid molecules according to the invention can encode an R1 protein from any desired plant of the genus Lolium; preferably, they encode a Lolium perenne R1 protein.

[0120] The nucleic acid molecules according to the invention can take the form of any desired nucleic acid molecules, in particular DNA or RNA molecules, for example cDNA, genomic DNA, mRNA and the like. They may be naturally occurring molecules or else molecules generated by recombinant or chemical synthetic methods. They may take the form of simplexes, which comprise either the coding or the noncoding strand, or duplexes.

[0121] The invention furthermore relates to vectors, in particular plasmids, cosmids, viruses, bacteriophages and other vectors conventionally used in genetic engineering which comprise the above-described nucleic acid molecules according to the invention.

[0122] In a preferred embodiment, the nucleic acid molecules which are present in the vectors are linked in sense orientation to regulatory elements which ensure expression in prokaryotic or eukaryotic cells. In this context, the term “expression” may refer to transcription or else to transcription and translation.

[0123] Expression of the nucleic acid molecules according to the invention in prokaryotic cells, for example in Escherichia coli, makes possible for example a more in-depth characterization of the biological activities of the encoded proteins. In addition, various types of mutations can be introduced into the nucleic acid molecules according to the invention by means of customary molecular-biological techniques (see, for example, Sambrook and Russell, loc. cit.), resulting in the synthesis of the proteins whose biological properties may have been altered. A possibility is the generation of deletion mutants, in which the consecutive deletions from the 5′ or from the 3′ end of the coding DNA sequence give rise to nucleic acid molecules which lead to the synthesis of correspondingly truncated proteins. Another possibility is the introduction of point mutations. The recombinant manipulation in prokaryotic cells can be carried out by methods known to the skilled worker (cf. Sambrook and Russell, loc. cit.).

[0124] Regulatory sequences for expression in prokaryotic organisms, for example E. coli, and in eukaryotic organisms have been described widely in the literature, in particular those for expression in yeast, such as, for example, Saccharomyces cerevisiae. An overview of various systems for expressing proteins in various host organisms is found, for example, in Duffaud et al. (Methods in Enzymology 153 (1987), 383-516) and in Bitter et al. (Methods in Enzymology 153 (1987), 516-544).

[0125] In a further embodiment, the invention relates to host cells, in particular prokaryotic or eukaryotic cells, which have been transformed with an above-described nucleic acid molecule or a vector, and to cells which are derived from such host cells and which comprise the above-described nucleic acid molecules or vectors. The host cells can be bacterial cells (for example E. coli) or fungal cells (for example yeast, in particular S. cerevisiae), or else plant or animal cells. In this context, the term “transformed” is understood as meaning that the cells according to the invention are genetically modified with a nucleic acid molecule according to the invention in as far as they comprise at least one nucleic acid molecule according to the invention in addition to their natural genome. The nucleic acid molecule according to the invention may be present in the cell in free form, if appropriate in the form of a self-replicating molecule, or may be present as a stable integration into the genome of the host cell. Preferably, the host cells are plant cells.

[0126] The present invention furthermore relates to methods for producing an R1 protein in which host cells according to the invention are grown under conditions which permit expression of the protein, and the protein is obtained from the culture, i.e. from the cells and/or the culture medium.

[0127] The present invention furthermore relates to a method for producing an R1 protein from plants of the genus Trifolium, Medicago or Lolium, the protein being produced in an in-vitro transcription and translation system using a nucleic acid molecule according to the invention. Such systems are known to the skilled worker.

[0128] The invention also relates to proteins which are encoded by the nucleic acid molecules according to the invention or which can be obtained by a method according to the invention.

[0129] The present invention furthermore relates to antibodies which specifically recognize a protein according to the invention. These antibodies may be for example monoclonal or polyclonal. They may also be fragments of antibodies which specifically recognize proteins according to the invention. Methods for preparing such antibodies or fragments are known to the skilled worker.

[0130] In particular, the present invention also relates to transgenic plant cells which comprise the nucleic acid molecules or vectors according to the invention. Preferably, the cells according to the invention are characterized in that the nucleic acid molecule according to the invention which has been introduced is stably integrated into the genome and under the control of a promoter which is active in plant cells, preferably a promoter which is heterologous with regard to the nucleic acid molecule.

[0131] A multiplicity of promoters is available for expressing a nucleic acid molecule according to the invention in plant cells. In principle, any promoter which is functional in the plants chosen for the transformation may be used. The promoter may be homologous or heterologous with regard to the plant species. Suitable examples are the cauliflower mosaic virus 35S promoter (Odell et al., Nature 313 (1985), 810-812), which ensures constitutive expression in all tissues of a plant, and the promoter is construct is described in WO 94/01571. Another example is the promoters of the polyubiquitine genes from maize. However, promoters which are activated only at a point in time which is determined by external influences may also be used (see, for example, WO 93/07279). In this context, promoters which may be of particular interest are promoters of heat shock proteins, which permit simple induction. Furthermore, those promoters which, in a particular tissue of the plant, lead to the expression of downstream sequences may be used (see, for example, Stockhaus et al., EMBO J. 8 (9), (1989), 2445-2451), for example the ST-LS1 promoter, which is only active in photosynthetically active tissue (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947). Other promoters which may be mentioned are those which are active in the starch-storing organs of plants to be transformed. These organs are, for example, the maize kernels in the case of maize and the tubers in the case of potatoes. The tuber-specific B33 promoter (Rocha-Sosa et al., EMBO J. 8 (1), (1989), 23-29) is an example which may be used for overexpressing the nucleic acid molecules according to the invention in potatoes. Seed-specific promoters have already been described for different plant species, for example the Vicia faba USP promoter, which ensures seed-specific expression in V. faba and other plants (Fiedler et al., Plant Mol. Biol. 22 (1993), 669-679; Bäumlein et al., Mol Gen. Genet. 225 (1991), 459-467). In maize, promoters which ensure specific expression in the endosperm of the maize kernels are, for example, promoters of the zein genes (Pedersen et al., Cell 29 (1982), 1015-1026; Quattrocchio et al., Plant Mol. Biol. 15 (1990), 81-93).

[0132] Thus, the expression of the nucleic acid molecules according to the invention in plant cells is possible.

[0133] The present invention thus also relates to a method for generating transgenic plant cells, comprising the introduction of a nucleic acid molecule or vector according to the invention into plant cells. Various plant transformation systems are available to the skilled worker; they have already been mentioned above.

[0134] When the nucleic acid molecules according to the invention are expressed in plants, it is possible, in principle, that the protein synthesized may be localized in any desired compartment of the plant cell. To achieve localization in the specific compartment, it may be necessary to link the coding region with DNA sequences which ensure localization in the compartment in question. Such sequences are known (see, for example, Braun, EMBO J. 11 (9), (1992), 3219-3227; Wolter, Proc. Natl. Acad. Sci. USA 85 (1988), 846-850; Sonnewald, Plant J. 1 (1), (1991), 95-106; Rocha-Sosa, EMBO J. 8 (1), (1989), 23-29).

[0135] The present invention thus also relates to transgenic plant cells which have been transformed with one or more nucleic acid molecule(s) according to the invention, and to transgenic plant cells which are derived from cells transformed thus. Such cells comprise one or more nucleic acid molecule(s) according to the invention, which is/are preferably linked to regulatory DNA elements which ensure transcription in plant cells, in particular with a promoter. Preferably, the promoter is heterologous with regard to the nucleic acid molecule. Such cells can be distinguished from naturally occurring plant cells by the fact that they comprise at least one nucleic acid molecule according to the invention in addition to any copies which may occur endogenously.

[0136] The transgenic plant cells can be regenerated into intact plants by techniques with which the skilled worker is familiar. The plants which can be obtained by regeneration of the transgenic plant cells according to the invention are likewise subject-matter of the present invention. Plants comprising the above-described transgenic plant cells are furthermore subject-matter of the invention. In principle, the transgenic plants may be plants of any desired plant species, i.e. both monocotyledonous and dicotyledonous plants, preferably fodder plants as defined above. The invention likewise relates to propagation material and harvested crops of the plants according to the invention, for example fruits, seeds, aerial parts, for example leaves, stalks and the like.

[0137] The nucleic acid molecules according to the invention or parts thereof can be employed in an above-described method according to the invention for generating fodder plants with an increased leaf starch content, preferably for generating plants of the genus Trifolium, Medicago and Lolium.

[0138] Moreover, the present invention relates to the use of the nucleic acid molecules according to the invention for identifying similar molecules which likewise encode an R1 protein. This can be done using techniques with which the skilled worker is familiar, for example by hybridization, screening gene libraries, amplification by means of suitable primers in a polymerase chain reaction, and the like.

[0139] Deposits

[0140] The following plasmids prepared and/or used for the purposes of the present invention were deposited at the Deutsche Sammlung von Mikroorganismen (DSM) in Brunswick, Federal Republic of Germany, which is recognized as international depository, in compliance with the provisions of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.

[0141] (Deposit number; Deposit date): Plasmid IR 116-156 (DSM 14707) (17 Dec. 2001) Plasmid IR 102-123 (DSM 14635) (16 Nov. 2001) Plasmid CF 19-49 (DSM 14633) (16 Nov. 2001)

[0142]FIG. 1 is a schematic representation of the vector piMs_R1 (Medicago sativa). A: Cauliflower mosaic virus CaMV 35S promoter, Franck et al., Cell 21 (1980), 285-294 A1: Partial R1 cDNA (˜1.9 kbp) from Medicago sativa (antisense orientation) A2: Terminator of the Agrobacterium tumefaciens octopine synthase gene, Gielen et al., EMBO J. 3, (1984) 835-846. B: Agrobacterium tumefaciens nopaline synthase promoter, Bevan et al., Nucl. Acids Res. 11 (1983), 369-385 B1: hph gene, Becker, Nucl. Acids Res. 18 (1990), 203 B2: Terminator of the nopaline synthase gene, Bevan et al., Nucl. Acids Res. 11 (1983), 369-385 LB: T-DNA left border, Gielen et al. (loc. cit.) RB: T-DNA right border, Gielen et al. (loc. cit.) KanR: nptIII gene, Trieu-Cout & Courvalin (1983) Gene 23: 331-341

[0143]FIG. 2 is a schematic representation of the vector piTr_R1 (Trifolium repens). A: Arabidopsis thaliana rbc-S promoter, De Almeida et al., Mol. Gen. Genet. 218 (1989), 78-86 A1: Partial R1 gene (˜1.1 kbp) from Trifolium repens (antisense orientation) A2: nos terminator, Depicker et al., J. Appl. Genet. 1 (1982), 561-573 B: CaMV 35S promoter, Franck et al. (loc. cit.) B1: bar gene, Thompson et al., EMBO J. 6 (1987), 2519-2523 B2: CaMV 35S terminator, Topfer et al., Nuc. Acids Res. 15 (1987), 5890 LB: T-DNA left border, Gielen et al. (loc. cit.) RB: T-DNA right border, Gielen et al. (loc. cit.) KanR: nptIII gene, Trieu-Cout & Courvalin, Gene 23 (1983), 331-341

[0144]FIG. 3 is a schematic representation of the vector piLp_R1 (Lolium perenne). A: CaMV 35S promoter, Franck et al. (loc. cit.) A1: Partial R1 fragment (1.07 kbp) from Lolium perenne (antisense orientation) A2: nos terminator, Depicker et al. (loc. cit.) B: Ubiquitin promoter and first intron from maize, Christensen et al., Plant Mol. Biol. 18 (1992), 675-689 B1: npt II gene, Garfinkel et al., Cell 27 (1981), 143-153 B2: CaMV 35S terminator (loc. cit.) LB: T-DNA left border (loc. cit.) RB: T-DNA right border (loc. cit.) KanR: nptIII gene (loc. cit.)

[0145] The examples which follow illustrate the invention.

EXAMPLE 1 Cloning Partial cDNA Sequences of the R1 Protein from Lolium perenne, Trifolium repens and Medicago sativa

[0146] The partial cDNA sequences of the R1 protein were cloned from Lolium perenne and Trifolium repens by PCR. (a) Nucleotide sequences of the primers used for PCR and RT-PCR R1-1: TACACCTGATATGCCAGATGTTC (SEQ ID NO: 9) R1-2: GGCCAYGGCATRCCAGA (Y = C, T; R = A, G) (SEQ ID NO: 10) Tr R1: AAGCCCGGGCAAGGAGGGTGAGGATATTGATGACA (SEQ ID NO: 11) Ms R1-2: CTACTCACGTTTGATTTGAAGTTGC (SEQ ID NO: 12) oligodT2: GAGAGAGTCGAGTTTTTTTTTTTTTTTTTTTTTTTTTT (SEQ ID NO: 13) Zm_R1-F3: GAGTGAACTTCAGCAATCAAGTTCTC (SEQ ID NO: 14)

[0147] (b) Isolation of a Genomic Fragment of the DNA of the R1 Gene from Trifolium repens

[0148] The following primer combinations were first used for amplifying parts of the genomic sequence of the Trifolium repens R1 gene: Size Primer 1 Primer 2 Template of the amplified fragment R1-1 R1-2 DNA from T. repens 4 kb

[0149] PCR Conditions: Kit: Platinum Taq Polymerase High Fidelity (Gibco) approx. 50 ng DNA from T. repens 400 μM primer R1-1 and 400 μM primer R1-2 final concentration 4 mM MgCl₂/200 μM dNTPs 2 min 94° C. denaturation 30 cycles: 20 sec 94° C. denaturation 30 sec 55° C. hybridization  1 min 70° C. elongation

[0150] The plasmid IR 93-123 contains the genomic fragment of the Trifolium repens R1 gene (blunt end cloning of the PCR amplificate by standard methods) in pBluescript SK (Stratagene), linearized using EcoRV.

[0151] (c) Cloning a Partial cDNA Fragment Encoding the Trifolium repens R1 Protein

[0152] A specific primer (TrR1) was synthesized using the sequence information of IR93-123. To amplify the cDNA fragment by means of RT-PCR, the following two primer combinations were used: Size Primer 1 Primer 2 Template of the amplified fragment Tr R1 R1-2 RNA from T. repens 0.5 kb Tr R1 ms R1-2 RNA from T. repens 1.1 kb

[0153] RT-PCR Conditions: Kit: One Step RT-PCR Kit from Qiagen approx. 200 ng of total RNA from T. repens leaves 500 nM primer TrR1 and 500 nM primer Ms R1-2 final concentration 5 mM MgCl₂/200 μM dNTPs 30 min 55° C. RT reaction 30 cycles:  15 sec 94° C. denaturation 15 min 95° C. Taq activation  30 sec 60° C. hybridization 1.5 min 72° C. elongation, final 10 min 72° C.

[0154] The plasmid IR 116-156 contains a 1.1 kb cDNA fragment of the Trifolium repens R1 (T/A cloning in pCR2.1 (Invitrogen), following the manufacturer's instructions). The sequence of the cDNA insertion is stated in SEQ ID NO: 1. To generate the plasmid piTr_R1, the approx. 1.07 kb SmaI/XbaI fragment from the plasmid IR 116-156 was cloned into the vector pi-TR downstream of the rbc_S promoter (in antisense orientation), using standard methods.

[0155] (d) Cloning a Partial cDNA Fragment Encoding the Lolium perenne R1 Protein

[0156] The following primer combination was used for amplifying the cDNA fragment by means of RT-PCR: Size of Primer 1 Primer 2 Template the amplified fragment R1-1 Oligo dT2 RNA from L. perenne 1.2 kb Zm_R1-F3 Oligo dT2 RNA from L. perenne 1.2 kb

[0157] RT-PCR Conditions Kit: One Step RT-PCR Kit from Qiagen approx. 200 ng total RNA from L. perenne leaves 500 nM primer R1-1 and 500 nM primer Oligo dT2 final concentration 5 mM MgCl₂/200 μM dNTPs 30 min 55° C. RT reaction  5 cycles:   15 sec 94° C. denaturation 15 min 95° C. Taq activation 25 cycles: 1.30 min 72° C. elongation   15 sec 94° C. denaturation   30 sec 60° C. Hybridization  1.5 min 72° C. elongation, final 10 min 72° C.

[0158] PCR Conditions for the Reamplification:

[0159] Kit: Platinum Taq DNA Polymerase High Fidelity (Gibco)

[0160] 50 ng PCR fragment from RT-PCR

[0161] 500 nM primer Zm_R1-F3 and 500 nM primer Oligo dT2

[0162] Final concentration 5 mM MgSO₄/200 μM dNTPs

[0163] 2 min 94° C. Taq activation

[0164] 30 cycles: 15 sec 94° C. denaturation 30 sec 60° C. hybridization 1.5 min 68° C. elongation, final 10 min 72° C.

[0165] The plasmid IR 102-123 contains a1.2 kb cDNA fragment of R1 from L. perenne (blunt-end cloning by standard methods into pBluescript SK⁺ (Stratagene) linearized with EcoRV. The sequence of the cDNA insertion is shown in SEQ ID NO: 5. To generate the plasmid piLp_R1, the approx. 1.1 kb XhoI fragment from the plasmid IR102-123 was cloned into the vector pi-Lp downstream of the CaMV 35S promoter (in antisense orientation), using standard methods.

[0166] (e) Isolation of a Partial cDNA for the Medicago Sativa R1 Protein

[0167] To screen a λZAPII cDNA library of Medicago sativa, 5×10⁵ recombinant phages were plated following the manufacturer's instructions (Stratagene). The phage DNA was transferred to Hybond N filters (Amersham) and immobilized thereon by means of a UV Stratalinker (Stratagene). Prehybridization was carried out for 4 hours at 42° C. (buffer: 5×SSC, 0.5% BSA, 5× Denhardt, 1% SDS, 40 mM phosphate buffer, pH 7.2, 100 mg/l herring sperm DNA, 25% formamide) and subsequently hybridized for 14 hours at the same temperature. The radiolabelled probe (Random Primed DNA Labeling Kit, Boehringer Mannheim, manufacturer's instructions) was the complete cDNA fragment encoding the potato R1 protein (see SEQ ID NO: 7). After hybridization, the filters were washed 3 times for 20 minutes with 3×SSC, 0.5% SDS at 50° C. and autoradiographed for 14 hours. The 6 plaques which showed the highest degree of hybridization were singled out by repeating the screening process three times, and the phages for in-vivo excision were used following the manufacturer's instructions (Stratagene). Plasmid DNA from resulting bacterial colonies was isolated by standard methods (Sambrook and Russell, 2000, loc. cit.) and subjected to DNA sequence analysis. One of the clones (pMs_R1.3) contained an approx. 1.85 kb cDNA fragment with the sequence shown in SEQ ID NO: 3.

[0168] (f) Preparation of a Vector for the Antisense Inhibition of the R1 Protein in Medicago sativa

[0169] To carry out the antisense inhibition, the complete cDNA fragment was excised from the plasmid pMs_R1.3 by means of the restriction enzymes Asp718 I and SmaI and ligated into the vector pBinAR-Hyg (CaMV 35S/ocs terminator cassette as EcoRI-HindIII fragment in pBIB-Hyg; Becker et al., Nucl. Acids Res. 18 (1990), 203). The resulting plasmid piMs_R1 is shown schematically in FIG. 1.

EXAMPLE 2 Plant Transformation

[0170] The transformation of Trifolium repens with the vector piTr_R1 was performed by the method described by Larkin et al. (Transgenic Research 5 (1996), 325-335). For the transformation by means of Agrobacterium tumefaciens (comprising plasmid piTr_R1), cotyledons of cultivar Haifa were used. In order to select the transformants, phosphinothricin was added to the B5PB medium in a concentration of 5 mg/l.

[0171] The transformation of Medicago sativa with the vector piMs_R1 was performed by the method described by Trinh et al. (Plant Cell Reports 17 (1998), 345-355). For the transformation by means of Agrobacterium tumefaciens (strain GV2260, comprising plasmid piMs_R1), leaf segments of Medicago sativa subspecies falcata (L.) PI.564263 were used.

[0172] After the coculture, Agrobacteria were suppressed by adding Ticarpen (500 mg/l) to the SHMab medium. In order to select the transformants, Hygromycin was added at a concentration of 10 mg/l.

[0173] The transformation of Lolium repens with the vector piLp_R1 was carried out by the method described by Altpeter et al. (Molecular Breeding 6 (2000), 519-528).

[0174] For the transformation, callus material from immature embryos of cultivar L6, which had previously been subcultured for five weeks, was used.

[0175] The transformants were selected by culturing the callus material which had been “bombarded” with plasmid piLp_R1 for two weeks on regeneration medium comprising paramomycin at a concentration of 100 mg/l.

EXAMPLE 3 Determination of the Starch Content in Leaf Material

[0176] (a) Sample Preparation:

[0177] Removal of the Soluble Sugars by Extraction with Ethanol:

[0178] Approx. 1 g of fresh leaf material from the transgenic plants generated as described in Example 2 was freeze-dried, weighed and subsequently homogenized to a fine powder using a Retsch ball mill. Approx. 50 mg of powdered leaf material (determination in duplicate) were weighed, 1 ml of 80% strength ethanol was added, the mixture was shaken vigorously, and the homogeneous dispersion was incubated for 1 h in a water bath at 80° C. After the dispersion had cooled to approx. 40° C., it was centrifuged for 5 min at 3 000 rpm (Minifuge RF, Heraeus). The supernatant was discarded. The leaf material was treated twice more with in each case 1 ml of 80% strength ethanol and incubated for in each case 20 min in a water bath at 80° C. After cooling and centrifuging (see above), all the supernatants were discarded.

[0179] (b) Starch Determination in a Microtitre Plate/Spectramax at 340 nm:

[0180] (Stärke Lebensmittelanalytik UV-Test), Boehringer Mannheim, Catalogue No.: 207748 (Amyloglucosidase, starch determination buffer, Glucose-6-phosphate dehydrogenase)

[0181] The sugar-free leaf material is treated with 400 microlitres 0.2 N KOH and homogenized by shaking vigorously. The homogenate is incubated for 1 h at 95° C. in a water bath. After cooling, 75 μl 1M acetic acid are added and the reaction mixture is mixed thoroughly. The mixture is centrifuged for 10 min at 4 000 rpm. 25 and 50 μl supernatant are introduced into a microtitre plate containing 50 μl amyloglucosidase (Boehringer Mannheim) and 25 or 50 μl, respectively, of Millipore water and digested for 1 h at 56° C. 196 μl starch determination buffer (Boehringer Mannheim) are introduced into another microtitre plate. To this there are added 4 (to 20) μl of the cooled starch digest. The ratio can be raised to up to 40 μl digest+160 μl starch determination buffer, depending on the glucose concentration. Measurement: shake, pre-read + 2 μl glucose-6-phosphate dehydrogenase (Boehringer Mannheim) incubation: 30 min at 37° C., measure

[0182] (c) The Starch Content was Calculated as Follows:

[0183] Measuring volume (200 μl)×extraction volume (4 750 μl)×amyloglucosidase digest volume (200 μl)×ΔOD/ε×1 000×sample measurement volume (4 μl . . . 40 μl)×sample digest volume (50 μl)×weight (g)×d(1)=concentration (μmol/g DW)

[0184] ε=6.3l×mmol⁻¹×cm⁻¹ (molar extinction coefficient of NADH at 340 nm)

[0185] DW=dry weight

[0186] The concentration in mg glucose/g fresh weight was calculated from the determined weights before and after freeze-drying and the molecular weight of glucose (162.1 g/mol—anhydride).

EXAMPLE 4

[0187] Leaf Starch Extraction Reagents Extraction buffer pH 7.3:  50 mM Na-MOPS MOPS = (3-[N-morpholino]propanesulphonic acid)   2 mM EDTA 0.5 mM beta-mercaptoethanol SDS 2% (Serva) ethanol 80% acetone

[0188] Starch Extraction

[0189] Using a Waring blender, the leaves of the plants were comminuted for approx. three minutes at the highest speed, using extraction buffer (8 ml per gram fresh weight). The mixture is subsequently filtered first through a kitchen strainer and then through a 125 μm filter. The solids are again homogenized in the Waring blender using extraction buffer (2 ml per gram fresh weight) and again filtered. After the second extraction, the solids are discarded. The filtrates are combined in a centrifuge bottle and centrifuged for 15 min at 5 500×g. The supernatant is discarded and the pellet is taken up in 2% SDS (8 ml per gram fresh weight). The suspension is filtered through a 30 μm filter with gentle stirring (starch passes through the filter) and then centrifuged for 15 min at 5 500×g. The supernatant is discarded and the pellet is washed three times with water (8 ml per gram fresh weight; resuspended and centrifuged as described above), and all the supernatants are discarded. Again, the pellet is taken up in water (8 ml per gram fresh weight) and the mixture is again filtered through a 30 μm filter, if possible without stirring.

[0190] The filtrate is subsequently centrifuged for 15 min at 5 500×g and the pellet is washed twice with 80% ethanol and once with acetone (in each case 0.5 ml per gram fresh weight; resuspended and centrifuged as described above).

[0191] After a final wash with water (0.5 ml per gram fresh weight), the pellet (leaf starch) is dried for at least 24 h in a lyophylizer.

[0192] Phosphate Determination Reagents: 0.7 N HCl:   2.9 ml 37% strength HCl/50 ml Buffer:    9 ml   1 M imidazole solution pH 7.2   225 μl   1 M MgCl₂    60 μl 0.5 M EDTA   150 μl  80 mM NADP 20.565 ml Millipore water  30.0 ml Enzyme: Glucose-6-phosphate dehydrogenase (from Leuconostoc mesenteroides) Roche, Catalogue No.: 165875, 1 000 U/1 ml, dilute 1:4 with buffer

[0193] Sample Preparation:

[0194] 100 mg leaf starch (weighed accurately) are weighed into a 2 ml Safe-Look Eppendorf tube and treated with 500 μl of 0.7 N HCl. During weighing, the water content of a further 100 mg of leaf starch is determined by means of a temperature-controlled balance.

[0195] The mixture is vortexed vigorously; this is followed by acid hydrolysis for 4 hours at 95° C. with shaking. After cooling, the mixture is centrifuged for 20 min at 13 000 rpm. All of the supernatant is transferred into a Spin Module Size 100 (Q.BIOgene) and filtered by briefly centrifuging.

[0196] 140 μl of hydrolysate are mixed with 1 260 μl of buffer in the Eppendorf tube, and two 700 μl aliquots are transferred into two quartz cuvettes (reference cuvette and sample cuvette). Enzymatic determination of glucose-6-phosphate is started by addition of 6 μl of enzyme to the sample cuvette. The measurement (increase in NADPH) is carried out at 340 nm using a UVIKON apparatus (Kontron).

[0197] Calculation: $\begin{matrix} \text{Calculation:} & \quad \\ {{\frac{{Measuring}\quad {{volume}\left( {700\quad {\mu l}} \right)} \times {extraction}\quad {{volume}\left( {500\mu \quad l} \right)} \times {delta}\quad {OD}}{ɛ \times {sample}\quad {{volume}\left( {70\mu \quad l} \right)} \times {{weight}^{*}({mg})} \times {d\left( {= 1} \right)}} = \begin{matrix} {{{C6P}\quad {concentration}}\quad} \\ {{{per}\quad {dry}\quad {leaf}\quad {starch}}\quad} \\ {n\quad {mol}\text{/}{mg}} \end{matrix}}\quad} & \quad \end{matrix}$

[0198] ε=6.3 l×mmol⁻¹×cm⁻¹ (molar extinction coefficient of NADPH at 340 nm)

[0199] d=path length of the cuvette

1 14 1 1141 DNA Trifolium repens CDS (1)..(1053) 1 aag gag ggt gag gat att gat gac aaa tca acc gac ctg aaa gaa gtt 48 Lys Glu Gly Glu Asp Ile Asp Asp Lys Ser Thr Asp Leu Lys Glu Val 1 5 10 15 ggt tct gtt cca act tta tct ttg gtc aga aag cag ttc agt ggt aga 96 Gly Ser Val Pro Thr Leu Ser Leu Val Arg Lys Gln Phe Ser Gly Arg 20 25 30 tat gct atc tca tct gaa gaa ttc act ggt gaa atg gtt gga gct aaa 144 Tyr Ala Ile Ser Ser Glu Glu Phe Thr Gly Glu Met Val Gly Ala Lys 35 40 45 cct cgt aat atc tct tac ttg aaa gga aag gtg cct tct tgg gta gga 192 Pro Arg Asn Ile Ser Tyr Leu Lys Gly Lys Val Pro Ser Trp Val Gly 50 55 60 att cct acc tcg gtt gcc tta cca ttt gga gtt ttt gaa cat gtt ctc 240 Ile Pro Thr Ser Val Ala Leu Pro Phe Gly Val Phe Glu His Val Leu 65 70 75 80 tct gat aag tca aat cag gtt gtg gct gag aag gtc aat att tta aaa 288 Ser Asp Lys Ser Asn Gln Val Val Ala Glu Lys Val Asn Ile Leu Lys 85 90 95 aag aag cta act gaa gga gac ttc agt gcc ctc aag gag att cgt gaa 336 Lys Lys Leu Thr Glu Gly Asp Phe Ser Ala Leu Lys Glu Ile Arg Glu 100 105 110 aca gtt tta gag ttg aat gct cca ccc aag ttg gta gag gag ttg aaa 384 Thr Val Leu Glu Leu Asn Ala Pro Pro Lys Leu Val Glu Glu Leu Lys 115 120 125 act aca atg aag ggt tct ggg atg cct tgg ccg ggt gat gaa ggt gaa 432 Thr Thr Met Lys Gly Ser Gly Met Pro Trp Pro Gly Asp Glu Gly Glu 130 135 140 caa cgc tgg gga cag gca tgg aag gct ata aaa aaa gtg tgg ggc tca 480 Gln Arg Trp Gly Gln Ala Trp Lys Ala Ile Lys Lys Val Trp Gly Ser 145 150 155 160 aag tgg aac gaa aga gcg tac ttc agc aca aga aaa gta aaa ctg gac 528 Lys Trp Asn Glu Arg Ala Tyr Phe Ser Thr Arg Lys Val Lys Leu Asp 165 170 175 cac gat tat ctt tcc atg tcc gtc ctt gta cag gaa gtg att aat gct 576 His Asp Tyr Leu Ser Met Ser Val Leu Val Gln Glu Val Ile Asn Ala 180 185 190 gac tat gcc ttt gtc atc cac aca acc aat ccg acc tct gga gac tct 624 Asp Tyr Ala Phe Val Ile His Thr Thr Asn Pro Thr Ser Gly Asp Ser 195 200 205 tca gaa ata tat aca gag gtt gta aag gga ctt ggt gaa aca ctg gtt 672 Ser Glu Ile Tyr Thr Glu Val Val Lys Gly Leu Gly Glu Thr Leu Val 210 215 220 gga gct tat ccc ggc cgt gct ttg agt ttt atc tgc aaa aaa cat gat 720 Gly Ala Tyr Pro Gly Arg Ala Leu Ser Phe Ile Cys Lys Lys His Asp 225 230 235 240 ttg aac tct cct cag gtc ttg ggt tat cct agc aaa cct atc ggg cta 768 Leu Asn Ser Pro Gln Val Leu Gly Tyr Pro Ser Lys Pro Ile Gly Leu 245 250 255 ttt ata aga cgg tca ata att ttt cgg tct gat tcc aat ggc gaa gat 816 Phe Ile Arg Arg Ser Ile Ile Phe Arg Ser Asp Ser Asn Gly Glu Asp 260 265 270 ctc gaa ggt tat gct ggt gct ggt ctt tat gac agt gtg cca atg gat 864 Leu Glu Gly Tyr Ala Gly Ala Gly Leu Tyr Asp Ser Val Pro Met Asp 275 280 285 gaa gaa gag aaa gtg gtg cta gat tac tca tca gat aaa ctg atg att 912 Glu Glu Glu Lys Val Val Leu Asp Tyr Ser Ser Asp Lys Leu Met Ile 290 295 300 gat ggt agt ttt cgc cag tct atc ttg tca agc att gcc cgt gca gga 960 Asp Gly Ser Phe Arg Gln Ser Ile Leu Ser Ser Ile Ala Arg Ala Gly 305 310 315 320 cat gca atc gaa gag ttg tat ggc act cct cag gac att gaa ggt gtc 1008 His Ala Ile Glu Glu Leu Tyr Gly Thr Pro Gln Asp Ile Glu Gly Val 325 330 335 atc aag gat gga aaa gtc tat gtt gtc cag acc aga cca caa gtg 1053 Ile Lys Asp Gly Lys Val Tyr Val Val Gln Thr Arg Pro Gln Val 340 345 350 taggtctcta gactcataac agataagctg cacatctcaa ctacgttcag caccatacta 1113 catgcaactt caaatcaaac gtgagtag 1141 2 351 PRT Trifolium repens 2 Lys Glu Gly Glu Asp Ile Asp Asp Lys Ser Thr Asp Leu Lys Glu Val 1 5 10 15 Gly Ser Val Pro Thr Leu Ser Leu Val Arg Lys Gln Phe Ser Gly Arg 20 25 30 Tyr Ala Ile Ser Ser Glu Glu Phe Thr Gly Glu Met Val Gly Ala Lys 35 40 45 Pro Arg Asn Ile Ser Tyr Leu Lys Gly Lys Val Pro Ser Trp Val Gly 50 55 60 Ile Pro Thr Ser Val Ala Leu Pro Phe Gly Val Phe Glu His Val Leu 65 70 75 80 Ser Asp Lys Ser Asn Gln Val Val Ala Glu Lys Val Asn Ile Leu Lys 85 90 95 Lys Lys Leu Thr Glu Gly Asp Phe Ser Ala Leu Lys Glu Ile Arg Glu 100 105 110 Thr Val Leu Glu Leu Asn Ala Pro Pro Lys Leu Val Glu Glu Leu Lys 115 120 125 Thr Thr Met Lys Gly Ser Gly Met Pro Trp Pro Gly Asp Glu Gly Glu 130 135 140 Gln Arg Trp Gly Gln Ala Trp Lys Ala Ile Lys Lys Val Trp Gly Ser 145 150 155 160 Lys Trp Asn Glu Arg Ala Tyr Phe Ser Thr Arg Lys Val Lys Leu Asp 165 170 175 His Asp Tyr Leu Ser Met Ser Val Leu Val Gln Glu Val Ile Asn Ala 180 185 190 Asp Tyr Ala Phe Val Ile His Thr Thr Asn Pro Thr Ser Gly Asp Ser 195 200 205 Ser Glu Ile Tyr Thr Glu Val Val Lys Gly Leu Gly Glu Thr Leu Val 210 215 220 Gly Ala Tyr Pro Gly Arg Ala Leu Ser Phe Ile Cys Lys Lys His Asp 225 230 235 240 Leu Asn Ser Pro Gln Val Leu Gly Tyr Pro Ser Lys Pro Ile Gly Leu 245 250 255 Phe Ile Arg Arg Ser Ile Ile Phe Arg Ser Asp Ser Asn Gly Glu Asp 260 265 270 Leu Glu Gly Tyr Ala Gly Ala Gly Leu Tyr Asp Ser Val Pro Met Asp 275 280 285 Glu Glu Glu Lys Val Val Leu Asp Tyr Ser Ser Asp Lys Leu Met Ile 290 295 300 Asp Gly Ser Phe Arg Gln Ser Ile Leu Ser Ser Ile Ala Arg Ala Gly 305 310 315 320 His Ala Ile Glu Glu Leu Tyr Gly Thr Pro Gln Asp Ile Glu Gly Val 325 330 335 Ile Lys Asp Gly Lys Val Tyr Val Val Gln Thr Arg Pro Gln Val 340 345 350 3 1855 DNA Medicago sativa CDS (3)..(1508) 3 ta ctt ggc gtg gaa aat tgg gct gtg gaa ata ttt act gaa gaa att 47 Leu Gly Val Glu Asn Trp Ala Val Glu Ile Phe Thr Glu Glu Ile 1 5 10 15 atc cgt gct gga tct gct gct tct ttg tct act ctt gta aat cga cta 95 Ile Arg Ala Gly Ser Ala Ala Ser Leu Ser Thr Leu Val Asn Arg Leu 20 25 30 gat cca gta ctc cga aag act gct aat ctt gga agt tgg cag gtt att 143 Asp Pro Val Leu Arg Lys Thr Ala Asn Leu Gly Ser Trp Gln Val Ile 35 40 45 agc cca gtt gaa acc gtt gga tat gtt gag gtt gtg gat gag ttg cta 191 Ser Pro Val Glu Thr Val Gly Tyr Val Glu Val Val Asp Glu Leu Leu 50 55 60 gct gtt caa aat aaa aca tat gag cga cct aca att ttg ata gcc aag 239 Ala Val Gln Asn Lys Thr Tyr Glu Arg Pro Thr Ile Leu Ile Ala Lys 65 70 75 agc gtg aga gga gag gag gaa att cca gac ggt aca gtt gct gtc ctg 287 Ser Val Arg Gly Glu Glu Glu Ile Pro Asp Gly Thr Val Ala Val Leu 80 85 90 95 aca cct gat atg cca gat gtt ctt tct cat gta tct gta cgg gca aga 335 Thr Pro Asp Met Pro Asp Val Leu Ser His Val Ser Val Arg Ala Arg 100 105 110 aat agc aag gtg tgt ttt gcg aca tgt ttt gat ccc aat atc ttt gct 383 Asn Ser Lys Val Cys Phe Ala Thr Cys Phe Asp Pro Asn Ile Phe Ala 115 120 125 gac ctc caa gca aat aaa gga aag ctc ttg cgg tta aag cct aca tca 431 Asp Leu Gln Ala Asn Lys Gly Lys Leu Leu Arg Leu Lys Pro Thr Ser 130 135 140 gct gag gtt gtt tat agt gag gtc aag gaa ggg gag aat att gat gac 479 Ala Glu Val Val Tyr Ser Glu Val Lys Glu Gly Glu Asn Ile Asp Asp 145 150 155 aaa tca acc gac ctg aaa gaa gtt gat tct att cca tct tta tct ttg 527 Lys Ser Thr Asp Leu Lys Glu Val Asp Ser Ile Pro Ser Leu Ser Leu 160 165 170 175 gtc aaa aag cag ttc agt ggt aga tat gct atc tct tct gaa gaa ttc 575 Val Lys Lys Gln Phe Ser Gly Arg Tyr Ala Ile Ser Ser Glu Glu Phe 180 185 190 act ggt gaa atg gtt gga gca aaa tcc cgt aat atc tcg tac tta aaa 623 Thr Gly Glu Met Val Gly Ala Lys Ser Arg Asn Ile Ser Tyr Leu Lys 195 200 205 gga aag gtg cct tct tgg gtt gga att cct acc tcg gtt gcc ata cca 671 Gly Lys Val Pro Ser Trp Val Gly Ile Pro Thr Ser Val Ala Ile Pro 210 215 220 ttt gga gtt ttt gaa cat gtt ctc tct gat aag tca aat cag gct gtg 719 Phe Gly Val Phe Glu His Val Leu Ser Asp Lys Ser Asn Gln Ala Val 225 230 235 gct gcg aag att gat gtt tta aaa aag aag tta act gaa gga gac ttc 767 Ala Ala Lys Ile Asp Val Leu Lys Lys Lys Leu Thr Glu Gly Asp Phe 240 245 250 255 ggt gcc ctc aag gag att cgt gaa aca gtt tta cag ttg aat gca cct 815 Gly Ala Leu Lys Glu Ile Arg Glu Thr Val Leu Gln Leu Asn Ala Pro 260 265 270 cct aag ttg ata gag gag ttg aaa act acg atg aag ggt tct ggg atg 863 Pro Lys Leu Ile Glu Glu Leu Lys Thr Thr Met Lys Gly Ser Gly Met 275 280 285 cct tgg ccg ggg gat gaa ggt gaa aaa cgc tgg gga cag gca tgg acg 911 Pro Trp Pro Gly Asp Glu Gly Glu Lys Arg Trp Gly Gln Ala Trp Thr 290 295 300 gct ata aaa aaa gtc tgg gga tca aag tgg aat gaa aga gcg tac ttc 959 Ala Ile Lys Lys Val Trp Gly Ser Lys Trp Asn Glu Arg Ala Tyr Phe 305 310 315 agc aca aga aaa gtg aag ctg gac cac gat tat ctt tcc atg tca gtc 1007 Ser Thr Arg Lys Val Lys Leu Asp His Asp Tyr Leu Ser Met Ser Val 320 325 330 335 ctt gta caa gaa gtg att aat gct gac tat gct ttt gtc atc cac aca 1055 Leu Val Gln Glu Val Ile Asn Ala Asp Tyr Ala Phe Val Ile His Thr 340 345 350 act aac ccg acc tct gga gac tct tca gaa ata tat act gag gtt gta 1103 Thr Asn Pro Thr Ser Gly Asp Ser Ser Glu Ile Tyr Thr Glu Val Val 355 360 365 aag gga ctg ggt gaa aca ctg gtt gga gct tat ccc ggc cgt gct ttg 1151 Lys Gly Leu Gly Glu Thr Leu Val Gly Ala Tyr Pro Gly Arg Ala Leu 370 375 380 agt ttt atc tgc aaa aaa caa gat ttg aac tct cct cag gtc ttg ggt 1199 Ser Phe Ile Cys Lys Lys Gln Asp Leu Asn Ser Pro Gln Val Leu Gly 385 390 395 tat cct agc aaa cct att ggg cta ttt ata aga cgg tca ata att ttt 1247 Tyr Pro Ser Lys Pro Ile Gly Leu Phe Ile Arg Arg Ser Ile Ile Phe 400 405 410 415 cga tct gat tcc aat ggt gaa gat ctc gaa ggt tac gct ggt gct ggt 1295 Arg Ser Asp Ser Asn Gly Glu Asp Leu Glu Gly Tyr Ala Gly Ala Gly 420 425 430 cta tat gac agt gtg cca atg gat gaa gaa gag aaa gtg gtg cta gat 1343 Leu Tyr Asp Ser Val Pro Met Asp Glu Glu Glu Lys Val Val Leu Asp 435 440 445 tac tcg tca gat aaa cta atg act aat agc agt ttt cgc cag tca atc 1391 Tyr Ser Ser Asp Lys Leu Met Thr Asn Ser Ser Phe Arg Gln Ser Ile 450 455 460 ttg tca agc att gcc agt gca gga aat gca atc gaa gag ttg tac ggc 1439 Leu Ser Ser Ile Ala Ser Ala Gly Asn Ala Ile Glu Glu Leu Tyr Gly 465 470 475 act cct cag gac att gaa ggt gtt gtc aag gat gga aaa atc tat gtt 1487 Thr Pro Gln Asp Ile Glu Gly Val Val Lys Asp Gly Lys Ile Tyr Val 480 485 490 495 gtc cag acc aga cca caa gtg taggtctcta gtctcattac agctaagcca 1538 Val Gln Thr Arg Pro Gln Val 500 cacatctcaa ctaaatttgt caccatattg cacggcaact tcaaatcaaa cgtgagtagc 1598 ttcttgcaaa attgagagct ttggtttgta cttttaatgt aactgatata tatgaaaata 1658 aagaataatt tacgtgtaac tatagaataa tgtaattatg gaaacatgca aatgtttcta 1718 ttttcccccc ctcttactat atgtaaaatt tgaatcctat tcttctcagt cgatgttgta 1778 tttttacttg atgtctactg tcaatggtta gataataaaa tacacagaaa tttattaaaa 1838 aaaaaaaaaa aaaaaaa 1855 4 502 PRT Medicago sativa 4 Leu Gly Val Glu Asn Trp Ala Val Glu Ile Phe Thr Glu Glu Ile Ile 1 5 10 15 Arg Ala Gly Ser Ala Ala Ser Leu Ser Thr Leu Val Asn Arg Leu Asp 20 25 30 Pro Val Leu Arg Lys Thr Ala Asn Leu Gly Ser Trp Gln Val Ile Ser 35 40 45 Pro Val Glu Thr Val Gly Tyr Val Glu Val Val Asp Glu Leu Leu Ala 50 55 60 Val Gln Asn Lys Thr Tyr Glu Arg Pro Thr Ile Leu Ile Ala Lys Ser 65 70 75 80 Val Arg Gly Glu Glu Glu Ile Pro Asp Gly Thr Val Ala Val Leu Thr 85 90 95 Pro Asp Met Pro Asp Val Leu Ser His Val Ser Val Arg Ala Arg Asn 100 105 110 Ser Lys Val Cys Phe Ala Thr Cys Phe Asp Pro Asn Ile Phe Ala Asp 115 120 125 Leu Gln Ala Asn Lys Gly Lys Leu Leu Arg Leu Lys Pro Thr Ser Ala 130 135 140 Glu Val Val Tyr Ser Glu Val Lys Glu Gly Glu Asn Ile Asp Asp Lys 145 150 155 160 Ser Thr Asp Leu Lys Glu Val Asp Ser Ile Pro Ser Leu Ser Leu Val 165 170 175 Lys Lys Gln Phe Ser Gly Arg Tyr Ala Ile Ser Ser Glu Glu Phe Thr 180 185 190 Gly Glu Met Val Gly Ala Lys Ser Arg Asn Ile Ser Tyr Leu Lys Gly 195 200 205 Lys Val Pro Ser Trp Val Gly Ile Pro Thr Ser Val Ala Ile Pro Phe 210 215 220 Gly Val Phe Glu His Val Leu Ser Asp Lys Ser Asn Gln Ala Val Ala 225 230 235 240 Ala Lys Ile Asp Val Leu Lys Lys Lys Leu Thr Glu Gly Asp Phe Gly 245 250 255 Ala Leu Lys Glu Ile Arg Glu Thr Val Leu Gln Leu Asn Ala Pro Pro 260 265 270 Lys Leu Ile Glu Glu Leu Lys Thr Thr Met Lys Gly Ser Gly Met Pro 275 280 285 Trp Pro Gly Asp Glu Gly Glu Lys Arg Trp Gly Gln Ala Trp Thr Ala 290 295 300 Ile Lys Lys Val Trp Gly Ser Lys Trp Asn Glu Arg Ala Tyr Phe Ser 305 310 315 320 Thr Arg Lys Val Lys Leu Asp His Asp Tyr Leu Ser Met Ser Val Leu 325 330 335 Val Gln Glu Val Ile Asn Ala Asp Tyr Ala Phe Val Ile His Thr Thr 340 345 350 Asn Pro Thr Ser Gly Asp Ser Ser Glu Ile Tyr Thr Glu Val Val Lys 355 360 365 Gly Leu Gly Glu Thr Leu Val Gly Ala Tyr Pro Gly Arg Ala Leu Ser 370 375 380 Phe Ile Cys Lys Lys Gln Asp Leu Asn Ser Pro Gln Val Leu Gly Tyr 385 390 395 400 Pro Ser Lys Pro Ile Gly Leu Phe Ile Arg Arg Ser Ile Ile Phe Arg 405 410 415 Ser Asp Ser Asn Gly Glu Asp Leu Glu Gly Tyr Ala Gly Ala Gly Leu 420 425 430 Tyr Asp Ser Val Pro Met Asp Glu Glu Glu Lys Val Val Leu Asp Tyr 435 440 445 Ser Ser Asp Lys Leu Met Thr Asn Ser Ser Phe Arg Gln Ser Ile Leu 450 455 460 Ser Ser Ile Ala Ser Ala Gly Asn Ala Ile Glu Glu Leu Tyr Gly Thr 465 470 475 480 Pro Gln Asp Ile Glu Gly Val Val Lys Asp Gly Lys Ile Tyr Val Val 485 490 495 Gln Thr Arg Pro Gln Val 500 5 1077 DNA Lolium perenne CDS (3)..(827) 5 tc gag aag gtc ttg cca gat gat acc aat aag gaa gta gca caa aac 47 Glu Lys Val Leu Pro Asp Asp Thr Asn Lys Glu Val Ala Gln Asn 1 5 10 15 ata cag atg ctg aag ggt aga ctt gat caa gat gat ttt agt gct ctt 95 Ile Gln Met Leu Lys Gly Arg Leu Asp Gln Asp Asp Phe Ser Ala Leu 20 25 30 gga gaa atg cgg aaa act gtt ctt gat tta act gct cca gct caa ctg 143 Gly Glu Met Arg Lys Thr Val Leu Asp Leu Thr Ala Pro Ala Gln Leu 35 40 45 gtt aca gag ctg aag gag aag atg cta agt tct gga atg ccc tgg cct 191 Val Thr Glu Leu Lys Glu Lys Met Leu Ser Ser Gly Met Pro Trp Pro 50 55 60 ggg gat gaa agt gac cag cgc tgg gag caa gca tgg atg gca att aaa 239 Gly Asp Glu Ser Asp Gln Arg Trp Glu Gln Ala Trp Met Ala Ile Lys 65 70 75 aag gtc tgg gca tca aaa tgg aac gaa aga gca tat ttt agc aca cgc 287 Lys Val Trp Ala Ser Lys Trp Asn Glu Arg Ala Tyr Phe Ser Thr Arg 80 85 90 95 aag gtg aag ctc gat cat gac tac ctt tcc atg gct gtt ctt gta cac 335 Lys Val Lys Leu Asp His Asp Tyr Leu Ser Met Ala Val Leu Val His 100 105 110 gaa att gtc aac gca gac tat gcc ttc gtc att cat act acg aac ccg 383 Glu Ile Val Asn Ala Asp Tyr Ala Phe Val Ile His Thr Thr Asn Pro 115 120 125 tca tct gga gat tct tcc gag ata tac gct gaa gtg gtg aaa gga ctt 431 Ser Ser Gly Asp Ser Ser Glu Ile Tyr Ala Glu Val Val Lys Gly Leu 130 135 140 gga gag aca ctt gtg gga gcc tat cct ggc cga gcc atg agc ttc gtg 479 Gly Glu Thr Leu Val Gly Ala Tyr Pro Gly Arg Ala Met Ser Phe Val 145 150 155 tgt aag aaa gat gac ctt gac tct ccc aag gta ctg ggt tac cca agt 527 Cys Lys Lys Asp Asp Leu Asp Ser Pro Lys Val Leu Gly Tyr Pro Ser 160 165 170 175 aag cca att ggt ctc ttc ata aag cga tca atc atc ttc cgt tca gat 575 Lys Pro Ile Gly Leu Phe Ile Lys Arg Ser Ile Ile Phe Arg Ser Asp 180 185 190 tct aat ggt gag gat ctg gaa ggt tat gct gga gca gga ctg tat gat 623 Ser Asn Gly Glu Asp Leu Glu Gly Tyr Ala Gly Ala Gly Leu Tyr Asp 195 200 205 agt gtc cct atg gat gag gaa gat caa gtt gtg ctc gac tac acg gcc 671 Ser Val Pro Met Asp Glu Glu Asp Gln Val Val Leu Asp Tyr Thr Ala 210 215 220 gac gct ctc atc acg gac tct gga ttc cga agc tca att ctc tca agc 719 Asp Ala Leu Ile Thr Asp Ser Gly Phe Arg Ser Ser Ile Leu Ser Ser 225 230 235 att gca cgg gct ggc cat gcc att gag gag ctc tac ggt tca cca cag 767 Ile Ala Arg Ala Gly His Ala Ile Glu Glu Leu Tyr Gly Ser Pro Gln 240 245 250 255 gac gtt gag gga gta gtc aag gat ggg aag atc tac gta gtc cag act 815 Asp Val Glu Gly Val Val Lys Asp Gly Lys Ile Tyr Val Val Gln Thr 260 265 270 aga cca cag atg taatacgtac ctataggcag ctcaagttgt agagtagtgg 867 Arg Pro Gln Met 275 gatatatgag cctttatggc atgcatagtt gtacaacaca tcatacatgt atgctcagaa 927 taaggtttaa ccatatactg tattttctgg agcttgtatt tgcaccaata aagtgtatgt 987 atcatgagga atatgaacct actaaaaaat tcagaggaaa catttttgag caataaaaaa 1047 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1077 6 275 PRT Lolium perenne 6 Glu Lys Val Leu Pro Asp Asp Thr Asn Lys Glu Val Ala Gln Asn Ile 1 5 10 15 Gln Met Leu Lys Gly Arg Leu Asp Gln Asp Asp Phe Ser Ala Leu Gly 20 25 30 Glu Met Arg Lys Thr Val Leu Asp Leu Thr Ala Pro Ala Gln Leu Val 35 40 45 Thr Glu Leu Lys Glu Lys Met Leu Ser Ser Gly Met Pro Trp Pro Gly 50 55 60 Asp Glu Ser Asp Gln Arg Trp Glu Gln Ala Trp Met Ala Ile Lys Lys 65 70 75 80 Val Trp Ala Ser Lys Trp Asn Glu Arg Ala Tyr Phe Ser Thr Arg Lys 85 90 95 Val Lys Leu Asp His Asp Tyr Leu Ser Met Ala Val Leu Val His Glu 100 105 110 Ile Val Asn Ala Asp Tyr Ala Phe Val Ile His Thr Thr Asn Pro Ser 115 120 125 Ser Gly Asp Ser Ser Glu Ile Tyr Ala Glu Val Val Lys Gly Leu Gly 130 135 140 Glu Thr Leu Val Gly Ala Tyr Pro Gly Arg Ala Met Ser Phe Val Cys 145 150 155 160 Lys Lys Asp Asp Leu Asp Ser Pro Lys Val Leu Gly Tyr Pro Ser Lys 165 170 175 Pro Ile Gly Leu Phe Ile Lys Arg Ser Ile Ile Phe Arg Ser Asp Ser 180 185 190 Asn Gly Glu Asp Leu Glu Gly Tyr Ala Gly Ala Gly Leu Tyr Asp Ser 195 200 205 Val Pro Met Asp Glu Glu Asp Gln Val Val Leu Asp Tyr Thr Ala Asp 210 215 220 Ala Leu Ile Thr Asp Ser Gly Phe Arg Ser Ser Ile Leu Ser Ser Ile 225 230 235 240 Ala Arg Ala Gly His Ala Ile Glu Glu Leu Tyr Gly Ser Pro Gln Asp 245 250 255 Val Glu Gly Val Val Lys Asp Gly Lys Ile Tyr Val Val Gln Thr Arg 260 265 270 Pro Gln Met 275 7 4826 DNA Solanum tuberosum CDS (104)..(4495) 7 atcttcatcg aatttctcga cgcttcttcg ctaatttcct cgttacttca ctagaaatcg 60 acgtttctag ctgaacttga gtgaattaag ccagtgggag gat atg agt aat tcc 115 Met Ser Asn Ser 1 tta ggg aat aac ttg ctg tac cag gga ttc cta acc tca aca gtg ttg 163 Leu Gly Asn Asn Leu Leu Tyr Gln Gly Phe Leu Thr Ser Thr Val Leu 5 10 15 20 gaa cat aaa agt aga atc agt cct cct tgt gtt gga ggc aat tct ttg 211 Glu His Lys Ser Arg Ile Ser Pro Pro Cys Val Gly Gly Asn Ser Leu 25 30 35 ttt caa caa caa gtg atc tcg aaa tca cct tta tca act gag ttt cga 259 Phe Gln Gln Gln Val Ile Ser Lys Ser Pro Leu Ser Thr Glu Phe Arg 40 45 50 ggt aac agg tta aag gtg cag aaa aag aaa ata cct atg gga aag aac 307 Gly Asn Arg Leu Lys Val Gln Lys Lys Lys Ile Pro Met Gly Lys Asn 55 60 65 cgt gct ttt tct agt tct cct cat gct gta ctt acc act gat acc tct 355 Arg Ala Phe Ser Ser Ser Pro His Ala Val Leu Thr Thr Asp Thr Ser 70 75 80 tct gag cta gca gaa aag ttc agt cta gaa ggg aat att gag cta cag 403 Ser Glu Leu Ala Glu Lys Phe Ser Leu Glu Gly Asn Ile Glu Leu Gln 85 90 95 100 gtt gat gtt agg cct ccc act tca ggt gat gtg tcc ttt gtg gat ttt 451 Val Asp Val Arg Pro Pro Thr Ser Gly Asp Val Ser Phe Val Asp Phe 105 110 115 caa gct aca aat ggt agt gat aaa ctg ttt ttg cac tgg ggg gca gta 499 Gln Ala Thr Asn Gly Ser Asp Lys Leu Phe Leu His Trp Gly Ala Val 120 125 130 aag ttc gga aaa gaa aca tgg tct ctt cct aat gat cgt cca gat ggg 547 Lys Phe Gly Lys Glu Thr Trp Ser Leu Pro Asn Asp Arg Pro Asp Gly 135 140 145 acc aaa gtg tac aag aac aaa gca ctt aga act cca ttt gtt aaa tct 595 Thr Lys Val Tyr Lys Asn Lys Ala Leu Arg Thr Pro Phe Val Lys Ser 150 155 160 ggc tct aac tcc atc ctg aga ctg gag ata cgg gac act gct atc gaa 643 Gly Ser Asn Ser Ile Leu Arg Leu Glu Ile Arg Asp Thr Ala Ile Glu 165 170 175 180 gct att gag ttt ctc ata tac gat gaa gcc tac gat aaa tgg ata aag 691 Ala Ile Glu Phe Leu Ile Tyr Asp Glu Ala Tyr Asp Lys Trp Ile Lys 185 190 195 aat aat ggt ggc aat ttt cgt gtc aaa ttg tca aga aaa gag ata cga 739 Asn Asn Gly Gly Asn Phe Arg Val Lys Leu Ser Arg Lys Glu Ile Arg 200 205 210 ggc cca gat gtt tca gtt cct gag gag ctt gta cag atc caa tca tat 787 Gly Pro Asp Val Ser Val Pro Glu Glu Leu Val Gln Ile Gln Ser Tyr 215 220 225 ttg agg tgg gag agg aag gga aaa cag aat tac acc cct gag aaa gag 835 Leu Arg Trp Glu Arg Lys Gly Lys Gln Asn Tyr Thr Pro Glu Lys Glu 230 235 240 aag gag gaa tat gag gct gct cga act gag cta cag gag gaa ata gct 883 Lys Glu Glu Tyr Glu Ala Ala Arg Thr Glu Leu Gln Glu Glu Ile Ala 245 250 255 260 cgt ggt gct tcc ata cag gac att cga gca agg cta aca aaa act aat 931 Arg Gly Ala Ser Ile Gln Asp Ile Arg Ala Arg Leu Thr Lys Thr Asn 265 270 275 gat aaa agt caa agc aaa gaa gag cct ctt cat gta aca aag agt gaa 979 Asp Lys Ser Gln Ser Lys Glu Glu Pro Leu His Val Thr Lys Ser Glu 280 285 290 ata cct gat gac ctt gcc caa gca caa gct tac att agg tgg gag aaa 1027 Ile Pro Asp Asp Leu Ala Gln Ala Gln Ala Tyr Ile Arg Trp Glu Lys 295 300 305 gca gga aag ccg aac tat cct cca gaa aag caa att gaa gaa ctc gaa 1075 Ala Gly Lys Pro Asn Tyr Pro Pro Glu Lys Gln Ile Glu Glu Leu Glu 310 315 320 gaa gca aga aga gaa ttg caa ctt gag ctt gag aaa ggc att acc ctt 1123 Glu Ala Arg Arg Glu Leu Gln Leu Glu Leu Glu Lys Gly Ile Thr Leu 325 330 335 340 gat gag ttg cgg aaa aag att aca aaa ggg gag ata aaa act aag gcg 1171 Asp Glu Leu Arg Lys Lys Ile Thr Lys Gly Glu Ile Lys Thr Lys Ala 345 350 355 gaa aag cac gtg aaa aga agc tct ttt gcc gtt gaa aga atc caa aga 1219 Glu Lys His Val Lys Arg Ser Ser Phe Ala Val Glu Arg Ile Gln Arg 360 365 370 aag aag aga gac ttt ggg cag ctt att aat aag tat cct tcc agt cct 1267 Lys Lys Arg Asp Phe Gly Gln Leu Ile Asn Lys Tyr Pro Ser Ser Pro 375 380 385 gca gta caa gta caa aag gtc ttg gaa gaa cca cca gcc tta tct aaa 1315 Ala Val Gln Val Gln Lys Val Leu Glu Glu Pro Pro Ala Leu Ser Lys 390 395 400 att aag ctg tat gcc aag gag aag gag gag cag att gat gat ccg atc 1363 Ile Lys Leu Tyr Ala Lys Glu Lys Glu Glu Gln Ile Asp Asp Pro Ile 405 410 415 420 ctt aat aaa aag atc ttt aag gtc gat gat ggg gag cta ctg gta ctg 1411 Leu Asn Lys Lys Ile Phe Lys Val Asp Asp Gly Glu Leu Leu Val Leu 425 430 435 gta gca aag tcc tct ggg aag aca aaa gta cat ata gct aca gat ctg 1459 Val Ala Lys Ser Ser Gly Lys Thr Lys Val His Ile Ala Thr Asp Leu 440 445 450 aat cag cca att act ctt cac tgg gca tta tcc aaa agt cgt gga gag 1507 Asn Gln Pro Ile Thr Leu His Trp Ala Leu Ser Lys Ser Arg Gly Glu 455 460 465 tgg atg gta cca cct tca agc ata ttg cct cct gga tca att att tta 1555 Trp Met Val Pro Pro Ser Ser Ile Leu Pro Pro Gly Ser Ile Ile Leu 470 475 480 gac aag gct gcc gaa aca cct ttt tcc gcc agt tct tct gat ggt cta 1603 Asp Lys Ala Ala Glu Thr Pro Phe Ser Ala Ser Ser Ser Asp Gly Leu 485 490 495 500 act tct aag gta caa tct ttg gat ata gta att gaa gat ggc aat ttt 1651 Thr Ser Lys Val Gln Ser Leu Asp Ile Val Ile Glu Asp Gly Asn Phe 505 510 515 gtg ggg atg cca ttt gtt ctt ttg tct ggt gaa aaa tgg att aag aac 1699 Val Gly Met Pro Phe Val Leu Leu Ser Gly Glu Lys Trp Ile Lys Asn 520 525 530 caa ggg tcg gat ttc tat gtt gac ttc agt gct gca tcc aaa tta gca 1747 Gln Gly Ser Asp Phe Tyr Val Asp Phe Ser Ala Ala Ser Lys Leu Ala 535 540 545 ctc aag gct gct ggg gat ggc agt gga act gca aag tct tta ctg gat 1795 Leu Lys Ala Ala Gly Asp Gly Ser Gly Thr Ala Lys Ser Leu Leu Asp 550 555 560 aaa ata gca gat atg gaa agt gag gct cag aag tca ttt atg cac cgg 1843 Lys Ile Ala Asp Met Glu Ser Glu Ala Gln Lys Ser Phe Met His Arg 565 570 575 580 ttt aat att gct gct gac ttg ata gaa gat gcc act agt gct ggt gaa 1891 Phe Asn Ile Ala Ala Asp Leu Ile Glu Asp Ala Thr Ser Ala Gly Glu 585 590 595 ctt ggt ttt act gga att ctt gta tgg atg agg ttc atg gct aca agg 1939 Leu Gly Phe Thr Gly Ile Leu Val Trp Met Arg Phe Met Ala Thr Arg 600 605 610 caa ctg ata tgg aac aaa aac tat aac gta aaa cca cgt gaa ata agc 1987 Gln Leu Ile Trp Asn Lys Asn Tyr Asn Val Lys Pro Arg Glu Ile Ser 615 620 625 aag gct cag gac aga ctt aca gac ttg ttg cag aat gct ttc acc agt 2035 Lys Ala Gln Asp Arg Leu Thr Asp Leu Leu Gln Asn Ala Phe Thr Ser 630 635 640 cac cct caa tac cgt gaa att ttg cgg atg att atg tca act gtt gga 2083 His Pro Gln Tyr Arg Glu Ile Leu Arg Met Ile Met Ser Thr Val Gly 645 650 655 660 cgt gga ggt gaa ggg gat gta gga cag cga att agg gat gaa att ttg 2131 Arg Gly Gly Glu Gly Asp Val Gly Gln Arg Ile Arg Asp Glu Ile Leu 665 670 675 gtc atc cag agg aaa aat gac tgc aag ggt ggt atg atg gaa gaa tgg 2179 Val Ile Gln Arg Lys Asn Asp Cys Lys Gly Gly Met Met Glu Glu Trp 680 685 690 cat cag aaa ttg cat aat aat act agt cct gat gat gtt gtg atc tgt 2227 His Gln Lys Leu His Asn Asn Thr Ser Pro Asp Asp Val Val Ile Cys 695 700 705 cag gca ttg att gac tac atc aag agt gat ttt gat ctt ggt gtt tat 2275 Gln Ala Leu Ile Asp Tyr Ile Lys Ser Asp Phe Asp Leu Gly Val Tyr 710 715 720 tgg aaa acc ctg aat gag aac gga ata aca aaa gag cgt ctt ttg agt 2323 Trp Lys Thr Leu Asn Glu Asn Gly Ile Thr Lys Glu Arg Leu Leu Ser 725 730 735 740 tat gac cgt gct atc cat tct gaa ccg aat ttt aga gga gat caa aag 2371 Tyr Asp Arg Ala Ile His Ser Glu Pro Asn Phe Arg Gly Asp Gln Lys 745 750 755 aat ggt ctt ttg cgt gat tta ggt cac tat atg aga aca ttg aag gct 2419 Asn Gly Leu Leu Arg Asp Leu Gly His Tyr Met Arg Thr Leu Lys Ala 760 765 770 gtt cat tca ggt gca gat ctt gag tct gct att gca aac tgc atg ggc 2467 Val His Ser Gly Ala Asp Leu Glu Ser Ala Ile Ala Asn Cys Met Gly 775 780 785 tac aaa act gag gga gaa ggc ttt atg gtt gga gtc cag ata aat cct 2515 Tyr Lys Thr Glu Gly Glu Gly Phe Met Val Gly Val Gln Ile Asn Pro 790 795 800 gta tca ggc ttg cca tct ggc ttt cag ggc ctc ctc cat ttt gtc tta 2563 Val Ser Gly Leu Pro Ser Gly Phe Gln Gly Leu Leu His Phe Val Leu 805 810 815 820 gac cat gtg gaa gat aaa aat gtg gaa act ctt ctt gag gga ttg cta 2611 Asp His Val Glu Asp Lys Asn Val Glu Thr Leu Leu Glu Gly Leu Leu 825 830 835 gag gct cgt gag gag ctt agg ccc ttg ctt ctc aaa cca aac aac cgt 2659 Glu Ala Arg Glu Glu Leu Arg Pro Leu Leu Leu Lys Pro Asn Asn Arg 840 845 850 cta aag gat ctg ctg ttt ttg gac ata gca ctt gat tct aca gtt aga 2707 Leu Lys Asp Leu Leu Phe Leu Asp Ile Ala Leu Asp Ser Thr Val Arg 855 860 865 aca gca gta gaa agg gga tat gaa gaa ttg aac aac gct aat cct gag 2755 Thr Ala Val Glu Arg Gly Tyr Glu Glu Leu Asn Asn Ala Asn Pro Glu 870 875 880 aaa atc atg tac ttc atc tcc ctc gtt ctt gaa aat ctc gca ctc tct 2803 Lys Ile Met Tyr Phe Ile Ser Leu Val Leu Glu Asn Leu Ala Leu Ser 885 890 895 900 gtg gac gat aat gaa gat ctt gtt tat tgc ttg aag gga tgg aat caa 2851 Val Asp Asp Asn Glu Asp Leu Val Tyr Cys Leu Lys Gly Trp Asn Gln 905 910 915 gct ctt tca atg tcc aat ggt gga gac aac cat tgg gct tta ttt gca 2899 Ala Leu Ser Met Ser Asn Gly Gly Asp Asn His Trp Ala Leu Phe Ala 920 925 930 aaa gct gta ctt gac aga atc cgt ctt gca ctt gca agc aag gca gag 2947 Lys Ala Val Leu Asp Arg Ile Arg Leu Ala Leu Ala Ser Lys Ala Glu 935 940 945 tgg tac cat cac tta ttg cag cca tct gcc gaa tat cta gga tca atc 2995 Trp Tyr His His Leu Leu Gln Pro Ser Ala Glu Tyr Leu Gly Ser Ile 950 955 960 ctt ggg gtg gac caa tgg gct ttg aac ata ttt act gaa gaa att ata 3043 Leu Gly Val Asp Gln Trp Ala Leu Asn Ile Phe Thr Glu Glu Ile Ile 965 970 975 980 cgt gct gga tca gca gct tca tta tcc tct ctt ctt aat aga ctc gat 3091 Arg Ala Gly Ser Ala Ala Ser Leu Ser Ser Leu Leu Asn Arg Leu Asp 985 990 995 ccc gtg ctt cgg aaa act gca aat cta gga agt tgg cag att atc 3136 Pro Val Leu Arg Lys Thr Ala Asn Leu Gly Ser Trp Gln Ile Ile 1000 1005 1010 agt cca gtt gaa gcc gtt gga tat gtt gtc gtt gtg gat gag ttg 3181 Ser Pro Val Glu Ala Val Gly Tyr Val Val Val Val Asp Glu Leu 1015 1020 1025 ctt tca gtt cag aat gaa atc tac aag aag ccc acg atc tta gta 3226 Leu Ser Val Gln Asn Glu Ile Tyr Lys Lys Pro Thr Ile Leu Val 1030 1035 1040 gca aac tct gtt aaa gga gag gag gaa att cct gat ggt gct gtt 3271 Ala Asn Ser Val Lys Gly Glu Glu Glu Ile Pro Asp Gly Ala Val 1045 1050 1055 gcc ctg ata aca cca gac atg cca gat gtt ctt tca cat gtt tct 3316 Ala Leu Ile Thr Pro Asp Met Pro Asp Val Leu Ser His Val Ser 1060 1065 1070 gtt cga gct aga aat ggg aag gtt tgc ttt gct aca tgc ttt gat 3361 Val Arg Ala Arg Asn Gly Lys Val Cys Phe Ala Thr Cys Phe Asp 1075 1080 1085 ccc aat ata ttg gct gac ctc caa gca aag gaa gga agg att ttg 3406 Pro Asn Ile Leu Ala Asp Leu Gln Ala Lys Glu Gly Arg Ile Leu 1090 1095 1100 ctc tta aag cct aca cct tca gac ata atc tat agt gag gtg aat 3451 Leu Leu Lys Pro Thr Pro Ser Asp Ile Ile Tyr Ser Glu Val Asn 1105 1110 1115 gag att gag ctc caa agt tca agt aac ttg gta gaa gct gaa act 3496 Glu Ile Glu Leu Gln Ser Ser Ser Asn Leu Val Glu Ala Glu Thr 1120 1125 1130 tca gca aca ctt aga ttg gtg aaa aag caa ttt ggt ggt tgt tac 3541 Ser Ala Thr Leu Arg Leu Val Lys Lys Gln Phe Gly Gly Cys Tyr 1135 1140 1145 gca ata tca gca gat gaa ttc aca agt gaa atg gtt gga gct aaa 3586 Ala Ile Ser Ala Asp Glu Phe Thr Ser Glu Met Val Gly Ala Lys 1150 1155 1160 tca cgt aat att gca tat ctg aaa gga aaa gtg cct tcc tcg gtg 3631 Ser Arg Asn Ile Ala Tyr Leu Lys Gly Lys Val Pro Ser Ser Val 1165 1170 1175 gga att cct acg tca gta gct ctt cca ttt gga gtc ttt gag aaa 3676 Gly Ile Pro Thr Ser Val Ala Leu Pro Phe Gly Val Phe Glu Lys 1180 1185 1190 gta ctt tca gac gac ata aat cag gga gtg gca aaa gag ttg caa 3721 Val Leu Ser Asp Asp Ile Asn Gln Gly Val Ala Lys Glu Leu Gln 1195 1200 1205 att ctg acg aaa aaa cta tct gaa gga gac ttc agc gct ctt ggt 3766 Ile Leu Thr Lys Lys Leu Ser Glu Gly Asp Phe Ser Ala Leu Gly 1210 1215 1220 gaa att cgc aca acg att tta gat ctt tca gca cca gct caa ttg 3811 Glu Ile Arg Thr Thr Ile Leu Asp Leu Ser Ala Pro Ala Gln Leu 1225 1230 1235 gtc aaa gag ctg aag gaa aag atg cag ggt tct ggc atg cct tgg 3856 Val Lys Glu Leu Lys Glu Lys Met Gln Gly Ser Gly Met Pro Trp 1240 1245 1250 cct ggt gat gaa ggt cca aag cgg tgg gaa caa gca tgg atg gcc 3901 Pro Gly Asp Glu Gly Pro Lys Arg Trp Glu Gln Ala Trp Met Ala 1255 1260 1265 ata aaa aag gtg tgg gct tca aaa tgg aat gag aga gca tac ttc 3946 Ile Lys Lys Val Trp Ala Ser Lys Trp Asn Glu Arg Ala Tyr Phe 1270 1275 1280 agc aca agg aag gtg aaa ctg gat cat gac tat ctg tgc atg gct 3991 Ser Thr Arg Lys Val Lys Leu Asp His Asp Tyr Leu Cys Met Ala 1285 1290 1295 gtc ctt gtt caa gaa ata ata aat gct gat tat gca ttt gtc att 4036 Val Leu Val Gln Glu Ile Ile Asn Ala Asp Tyr Ala Phe Val Ile 1300 1305 1310 cac aca acc aac cca tct tcc gga gac gac tca gaa ata tat gcc 4081 His Thr Thr Asn Pro Ser Ser Gly Asp Asp Ser Glu Ile Tyr Ala 1315 1320 1325 gag gtg gtc agg ggc ctt ggg gaa aca ctt gtt gga gct tac cca 4126 Glu Val Val Arg Gly Leu Gly Glu Thr Leu Val Gly Ala Tyr Pro 1330 1335 1340 gga cgt gct ttg agt ttt atc tgc aag aaa aag gat ctc aac tct 4171 Gly Arg Ala Leu Ser Phe Ile Cys Lys Lys Lys Asp Leu Asn Ser 1345 1350 1355 cct caa gtg tta ggt tac cca agc aaa ccg atc ggc ctt ttc ata 4216 Pro Gln Val Leu Gly Tyr Pro Ser Lys Pro Ile Gly Leu Phe Ile 1360 1365 1370 aaa aga tct atc atc ttc cga tct gat tcc aat ggg gaa gat ttg 4261 Lys Arg Ser Ile Ile Phe Arg Ser Asp Ser Asn Gly Glu Asp Leu 1375 1380 1385 gaa ggt tat gcc ggt gct ggc ctc tac gac agt gta cca atg gat 4306 Glu Gly Tyr Ala Gly Ala Gly Leu Tyr Asp Ser Val Pro Met Asp 1390 1395 1400 gag gag gaa aaa gtt gta att gat tac tct tcc gac cca ttg ata 4351 Glu Glu Glu Lys Val Val Ile Asp Tyr Ser Ser Asp Pro Leu Ile 1405 1410 1415 act gat ggt aac ttc cgc cag aca atc ctg tcc aac att gct cgt 4396 Thr Asp Gly Asn Phe Arg Gln Thr Ile Leu Ser Asn Ile Ala Arg 1420 1425 1430 gct gga cat gct atc gag gag cta tat ggc tct cct caa gac atc 4441 Ala Gly His Ala Ile Glu Glu Leu Tyr Gly Ser Pro Gln Asp Ile 1435 1440 1445 gag ggt gta gtg agg gat gga aag att tat gtc gtt cag aca aga 4486 Glu Gly Val Val Arg Asp Gly Lys Ile Tyr Val Val Gln Thr Arg 1450 1455 1460 cct cag atg tgatcatatt ctcgttgtat gttgttcaga gaagaccata 4535 Pro Gln Met gatgtgatca tattctcatg gtatcagatc tgtgaccact tacctcccat gaagttgcct 4595 gtatgattat acgtgatcca aagccatcac atcatgttca ccttcagcta ttggaggaga 4655 agtgagaagt aggaattgca atatgaggaa taataagaaa aactttgtag aagttaaatt 4715 agctgggtat gatataggga gaaatgtgta aacattgtac tatatatagt atacacacgc 4775 attatgtatt tgcattatgc actgaataat atcgcagcat caaagaagaa a 4826 8 1464 PRT Solanum tuberosum 8 Met Ser Asn Ser Leu Gly Asn Asn Leu Leu Tyr Gln Gly Phe Leu Thr 1 5 10 15 Ser Thr Val Leu Glu His Lys Ser Arg Ile Ser Pro Pro Cys Val Gly 20 25 30 Gly Asn Ser Leu Phe Gln Gln Gln Val Ile Ser Lys Ser Pro Leu Ser 35 40 45 Thr Glu Phe Arg Gly Asn Arg Leu Lys Val Gln Lys Lys Lys Ile Pro 50 55 60 Met Gly Lys Asn Arg Ala Phe Ser Ser Ser Pro His Ala Val Leu Thr 65 70 75 80 Thr Asp Thr Ser Ser Glu Leu Ala Glu Lys Phe Ser Leu Glu Gly Asn 85 90 95 Ile Glu Leu Gln Val Asp Val Arg Pro Pro Thr Ser Gly Asp Val Ser 100 105 110 Phe Val Asp Phe Gln Ala Thr Asn Gly Ser Asp Lys Leu Phe Leu His 115 120 125 Trp Gly Ala Val Lys Phe Gly Lys Glu Thr Trp Ser Leu Pro Asn Asp 130 135 140 Arg Pro Asp Gly Thr Lys Val Tyr Lys Asn Lys Ala Leu Arg Thr Pro 145 150 155 160 Phe Val Lys Ser Gly Ser Asn Ser Ile Leu Arg Leu Glu Ile Arg Asp 165 170 175 Thr Ala Ile Glu Ala Ile Glu Phe Leu Ile Tyr Asp Glu Ala Tyr Asp 180 185 190 Lys Trp Ile Lys Asn Asn Gly Gly Asn Phe Arg Val Lys Leu Ser Arg 195 200 205 Lys Glu Ile Arg Gly Pro Asp Val Ser Val Pro Glu Glu Leu Val Gln 210 215 220 Ile Gln Ser Tyr Leu Arg Trp Glu Arg Lys Gly Lys Gln Asn Tyr Thr 225 230 235 240 Pro Glu Lys Glu Lys Glu Glu Tyr Glu Ala Ala Arg Thr Glu Leu Gln 245 250 255 Glu Glu Ile Ala Arg Gly Ala Ser Ile Gln Asp Ile Arg Ala Arg Leu 260 265 270 Thr Lys Thr Asn Asp Lys Ser Gln Ser Lys Glu Glu Pro Leu His Val 275 280 285 Thr Lys Ser Glu Ile Pro Asp Asp Leu Ala Gln Ala Gln Ala Tyr Ile 290 295 300 Arg Trp Glu Lys Ala Gly Lys Pro Asn Tyr Pro Pro Glu Lys Gln Ile 305 310 315 320 Glu Glu Leu Glu Glu Ala Arg Arg Glu Leu Gln Leu Glu Leu Glu Lys 325 330 335 Gly Ile Thr Leu Asp Glu Leu Arg Lys Lys Ile Thr Lys Gly Glu Ile 340 345 350 Lys Thr Lys Ala Glu Lys His Val Lys Arg Ser Ser Phe Ala Val Glu 355 360 365 Arg Ile Gln Arg Lys Lys Arg Asp Phe Gly Gln Leu Ile Asn Lys Tyr 370 375 380 Pro Ser Ser Pro Ala Val Gln Val Gln Lys Val Leu Glu Glu Pro Pro 385 390 395 400 Ala Leu Ser Lys Ile Lys Leu Tyr Ala Lys Glu Lys Glu Glu Gln Ile 405 410 415 Asp Asp Pro Ile Leu Asn Lys Lys Ile Phe Lys Val Asp Asp Gly Glu 420 425 430 Leu Leu Val Leu Val Ala Lys Ser Ser Gly Lys Thr Lys Val His Ile 435 440 445 Ala Thr Asp Leu Asn Gln Pro Ile Thr Leu His Trp Ala Leu Ser Lys 450 455 460 Ser Arg Gly Glu Trp Met Val Pro Pro Ser Ser Ile Leu Pro Pro Gly 465 470 475 480 Ser Ile Ile Leu Asp Lys Ala Ala Glu Thr Pro Phe Ser Ala Ser Ser 485 490 495 Ser Asp Gly Leu Thr Ser Lys Val Gln Ser Leu Asp Ile Val Ile Glu 500 505 510 Asp Gly Asn Phe Val Gly Met Pro Phe Val Leu Leu Ser Gly Glu Lys 515 520 525 Trp Ile Lys Asn Gln Gly Ser Asp Phe Tyr Val Asp Phe Ser Ala Ala 530 535 540 Ser Lys Leu Ala Leu Lys Ala Ala Gly Asp Gly Ser Gly Thr Ala Lys 545 550 555 560 Ser Leu Leu Asp Lys Ile Ala Asp Met Glu Ser Glu Ala Gln Lys Ser 565 570 575 Phe Met His Arg Phe Asn Ile Ala Ala Asp Leu Ile Glu Asp Ala Thr 580 585 590 Ser Ala Gly Glu Leu Gly Phe Thr Gly Ile Leu Val Trp Met Arg Phe 595 600 605 Met Ala Thr Arg Gln Leu Ile Trp Asn Lys Asn Tyr Asn Val Lys Pro 610 615 620 Arg Glu Ile Ser Lys Ala Gln Asp Arg Leu Thr Asp Leu Leu Gln Asn 625 630 635 640 Ala Phe Thr Ser His Pro Gln Tyr Arg Glu Ile Leu Arg Met Ile Met 645 650 655 Ser Thr Val Gly Arg Gly Gly Glu Gly Asp Val Gly Gln Arg Ile Arg 660 665 670 Asp Glu Ile Leu Val Ile Gln Arg Lys Asn Asp Cys Lys Gly Gly Met 675 680 685 Met Glu Glu Trp His Gln Lys Leu His Asn Asn Thr Ser Pro Asp Asp 690 695 700 Val Val Ile Cys Gln Ala Leu Ile Asp Tyr Ile Lys Ser Asp Phe Asp 705 710 715 720 Leu Gly Val Tyr Trp Lys Thr Leu Asn Glu Asn Gly Ile Thr Lys Glu 725 730 735 Arg Leu Leu Ser Tyr Asp Arg Ala Ile His Ser Glu Pro Asn Phe Arg 740 745 750 Gly Asp Gln Lys Asn Gly Leu Leu Arg Asp Leu Gly His Tyr Met Arg 755 760 765 Thr Leu Lys Ala Val His Ser Gly Ala Asp Leu Glu Ser Ala Ile Ala 770 775 780 Asn Cys Met Gly Tyr Lys Thr Glu Gly Glu Gly Phe Met Val Gly Val 785 790 795 800 Gln Ile Asn Pro Val Ser Gly Leu Pro Ser Gly Phe Gln Gly Leu Leu 805 810 815 His Phe Val Leu Asp His Val Glu Asp Lys Asn Val Glu Thr Leu Leu 820 825 830 Glu Gly Leu Leu Glu Ala Arg Glu Glu Leu Arg Pro Leu Leu Leu Lys 835 840 845 Pro Asn Asn Arg Leu Lys Asp Leu Leu Phe Leu Asp Ile Ala Leu Asp 850 855 860 Ser Thr Val Arg Thr Ala Val Glu Arg Gly Tyr Glu Glu Leu Asn Asn 865 870 875 880 Ala Asn Pro Glu Lys Ile Met Tyr Phe Ile Ser Leu Val Leu Glu Asn 885 890 895 Leu Ala Leu Ser Val Asp Asp Asn Glu Asp Leu Val Tyr Cys Leu Lys 900 905 910 Gly Trp Asn Gln Ala Leu Ser Met Ser Asn Gly Gly Asp Asn His Trp 915 920 925 Ala Leu Phe Ala Lys Ala Val Leu Asp Arg Ile Arg Leu Ala Leu Ala 930 935 940 Ser Lys Ala Glu Trp Tyr His His Leu Leu Gln Pro Ser Ala Glu Tyr 945 950 955 960 Leu Gly Ser Ile Leu Gly Val Asp Gln Trp Ala Leu Asn Ile Phe Thr 965 970 975 Glu Glu Ile Ile Arg Ala Gly Ser Ala Ala Ser Leu Ser Ser Leu Leu 980 985 990 Asn Arg Leu Asp Pro Val Leu Arg Lys Thr Ala Asn Leu Gly Ser Trp 995 1000 1005 Gln Ile Ile Ser Pro Val Glu Ala Val Gly Tyr Val Val Val Val 1010 1015 1020 Asp Glu Leu Leu Ser Val Gln Asn Glu Ile Tyr Lys Lys Pro Thr 1025 1030 1035 Ile Leu Val Ala Asn Ser Val Lys Gly Glu Glu Glu Ile Pro Asp 1040 1045 1050 Gly Ala Val Ala Leu Ile Thr Pro Asp Met Pro Asp Val Leu Ser 1055 1060 1065 His Val Ser Val Arg Ala Arg Asn Gly Lys Val Cys Phe Ala Thr 1070 1075 1080 Cys Phe Asp Pro Asn Ile Leu Ala Asp Leu Gln Ala Lys Glu Gly 1085 1090 1095 Arg Ile Leu Leu Leu Lys Pro Thr Pro Ser Asp Ile Ile Tyr Ser 1100 1105 1110 Glu Val Asn Glu Ile Glu Leu Gln Ser Ser Ser Asn Leu Val Glu 1115 1120 1125 Ala Glu Thr Ser Ala Thr Leu Arg Leu Val Lys Lys Gln Phe Gly 1130 1135 1140 Gly Cys Tyr Ala Ile Ser Ala Asp Glu Phe Thr Ser Glu Met Val 1145 1150 1155 Gly Ala Lys Ser Arg Asn Ile Ala Tyr Leu Lys Gly Lys Val Pro 1160 1165 1170 Ser Ser Val Gly Ile Pro Thr Ser Val Ala Leu Pro Phe Gly Val 1175 1180 1185 Phe Glu Lys Val Leu Ser Asp Asp Ile Asn Gln Gly Val Ala Lys 1190 1195 1200 Glu Leu Gln Ile Leu Thr Lys Lys Leu Ser Glu Gly Asp Phe Ser 1205 1210 1215 Ala Leu Gly Glu Ile Arg Thr Thr Ile Leu Asp Leu Ser Ala Pro 1220 1225 1230 Ala Gln Leu Val Lys Glu Leu Lys Glu Lys Met Gln Gly Ser Gly 1235 1240 1245 Met Pro Trp Pro Gly Asp Glu Gly Pro Lys Arg Trp Glu Gln Ala 1250 1255 1260 Trp Met Ala Ile Lys Lys Val Trp Ala Ser Lys Trp Asn Glu Arg 1265 1270 1275 Ala Tyr Phe Ser Thr Arg Lys Val Lys Leu Asp His Asp Tyr Leu 1280 1285 1290 Cys Met Ala Val Leu Val Gln Glu Ile Ile Asn Ala Asp Tyr Ala 1295 1300 1305 Phe Val Ile His Thr Thr Asn Pro Ser Ser Gly Asp Asp Ser Glu 1310 1315 1320 Ile Tyr Ala Glu Val Val Arg Gly Leu Gly Glu Thr Leu Val Gly 1325 1330 1335 Ala Tyr Pro Gly Arg Ala Leu Ser Phe Ile Cys Lys Lys Lys Asp 1340 1345 1350 Leu Asn Ser Pro Gln Val Leu Gly Tyr Pro Ser Lys Pro Ile Gly 1355 1360 1365 Leu Phe Ile Lys Arg Ser Ile Ile Phe Arg Ser Asp Ser Asn Gly 1370 1375 1380 Glu Asp Leu Glu Gly Tyr Ala Gly Ala Gly Leu Tyr Asp Ser Val 1385 1390 1395 Pro Met Asp Glu Glu Glu Lys Val Val Ile Asp Tyr Ser Ser Asp 1400 1405 1410 Pro Leu Ile Thr Asp Gly Asn Phe Arg Gln Thr Ile Leu Ser Asn 1415 1420 1425 Ile Ala Arg Ala Gly His Ala Ile Glu Glu Leu Tyr Gly Ser Pro 1430 1435 1440 Gln Asp Ile Glu Gly Val Val Arg Asp Gly Lys Ile Tyr Val Val 1445 1450 1455 Gln Thr Arg Pro Gln Met 1460 9 23 DNA Artificial sequence Oligonucleotide 9 tacacctgat atgccagatg ttc 23 10 17 DNA Artificial sequence Oligonucleotide 10 ggccayggca trccaga 17 11 35 DNA Artificial sequence Oligonucleotide 11 aagcccgggc aaggagggtg aggatattga tgaca 35 12 25 DNA Artificial sequence Oligonucleotide 12 ctactcacgt ttgatttgaa gttgc 25 13 38 DNA Artificial sequence Oligonucleotide 13 gagagactcg agtttttttt tttttttttt tttttttt 38 14 26 DNA Artificial sequence Oligonucleotide 14 gagtgaactt cagcaatcaa gttctc 26 

1. Method for generating a transgenic fodder plant with an increased leaf starch content in comparison to corresponding wild-type plants, where (a) a cell of a fodder plant is genetically modified by introducing a foreign nucleic acid molecule whose presence or expression leads to a reduced activity of an R1 protein which occurs endogenously in the plant cell; (b) a plant is regenerated from the cell generated in step (a); (c) if appropriate, further plants are generated starting from the plant generated in step (b); and (d) the foreign nucleic acid molecule according to step (a) is selected from DNA molecules which encode at least one antisense RNA which brings about reduced expression of endogenous genes which encode R1 proteins.
 2. Method for generating a transgenic fodder plant with an increased leaf starch content in comparison to corresponding wild-type plants, where (a) a cell of a fodder plant is genetically modified by introducing a foreign nucleic acid molecule whose presence or expression leads to a reduced activity of an R1 protein which occurs endogenously in the plant cell; (b) a plant is regenerated from the cell generated in step (a); (c) if appropriate, further plants are generated starting from the plant generated in step (b); and (d) the foreign nucleic acid molecule according to step (a) is selected from DNA molecules which, via a cosuppression effect, lead to reduced expression of endogenous genes which encode R1 proteins.
 3. Method for generating a transgenic fodder plant with an increased leaf starch content in comparison to corresponding wild-type plants, where (a) a cell of a fodder plant is genetically modified by introducing a foreign nucleic acid molecule whose presence or expression leads to a reduced activity of an R1 protein which occurs endogenously in the plant cell; (b) a plant is regenerated from the cell generated in step (a); (c) if appropriate, further plants are generated starting from the plant generated in step (b); and (d) the foreign nucleic acid molecule according to step (a) is selected from DNA molecules which encode at least one ribozyme which specifically cleaves transcripts of endogenous genes which encode R1 proteins.
 4. Method for generating a transgenic fodder plant with an increased leaf starch content in comparison to corresponding wild-type plants, where (a) a cell of a fodder plant is genetically modified by introducing a foreign nucleic acid molecule whose presence or expression leads to a reduced activity of an R1 protein which occurs endogenously in the plant cell; (b) a plant is regenerated from the cell generated in step (a); (c) if appropriate, further plants are generated starting from the plant generated in step (b); and (d) the foreign nucleic acid molecule according to step (a) is selected from nucleic acid molecules which, in the event of in vivo mutagenesis, lead to a mutation or an insertion of a heterologous sequence in endogenous genes which encode R1 proteins, the mutation or insertion leading to reduced expression of genes encoding R1 proteins, or to a reduced synthesis of active R1 proteins.
 5. Method for generating a transgenic fodder plant with an increased leaf starch content in comparison to corresponding wild-type plants, where (a) a cell of a fodder plant is genetically modified by introducing a foreign nucleic acid molecule whose presence or expression leads to a reduced activity of an R1 protein which occurs endogenously in the plant cell; (b) a plant is regenerated from the cell generated in step (a); (c) if appropriate, further plants are generated starting from the plant generated in step (b); and (d) the foreign nucleic acid molecule according to step (a) is selected from DNA molecules which simultaneously encode at least one antisense RNA and at least one sense RNA, where the antisense RNA and the sense RNA form a RNA duplex which brings about a reduced expression of endogenous genes which encode R1 proteins.
 6. Method according to claim 1, where the reduced activity of the R1 protein means an amount of R1 protein which is reduced by at least 50% in comparison with corresponding cells which have not been genetically modified.
 7. Method according to claim 1, where the fodder plant is a plant of the genus Trifolium.
 8. Method according to claim 1, where the fodder plant is a plant of the genus Medicago.
 9. Method according to claim 1, where the fodder plant is a plant of the genus Lolium.
 10. Transgenic fodder plant which has an increased leaf starch content and which can be obtained by a method for generating a transgenic fodder plant with an increased leaf starch content in comparison to corresponding wild-type plants, where (a) a cell of a fodder plant is genetically modified by introducing a foreign nucleic acid molecule whose presence or expression leads to a reduced activity of an R1 protein which occurs endogenously in the plant cell; (b) a plant is regenerated from the cell generated in step (a); (c) if appropriate, further plants are generated starting from the plant generated in step (b); and (d) the foreign nucleic acid molecule according to step (a) is selected from DNA molecules which encode at least one antisense RNA which brings about reduced expression of endogenous genes which encode R1 proteins.
 11. Transgenic fodder plant which has an increased leaf starch content and which can be obtained by a method for generating a transgenic fodder plant with an increased leaf starch content in comparison to corresponding wild-type plants, where (a) a cell of a fodder plant is genetically modified by introducing a foreign nucleic acid molecule whose presence or expression leads to a reduced activity of an R1 protein which occurs endogenously in the plant cell; (b) a plant is regenerated from the cell generated in step (a); (c) if appropriate, further plants are generated starting from the plant generated in step (b); and (d) the foreign nucleic acid molecule according to step (a) is selected from DNA molecules which, via a cosuppression effect, lead to reduced expression of endogenous genes which encode R1 proteins.
 12. Transgenic fodder plant which has an increased leaf starch content and which can be obtained by a method for generating a transgenic fodder plant with an increased leaf starch content in comparison to corresponding wild-type plants, where (a) a cell of a fodder plant is genetically modified by introducing a foreign nucleic acid molecule whose presence or expression leads to a reduced activity of an R1 protein which occurs endogenously in the plant cell; (b) a plant is regenerated from the cell generated in step (a); (c) if appropriate, further plants are generated starting from the plant generated in step (b); and (d) the foreign nucleic acid molecule according to step (a) is selected from DNA molecules which encode at least one ribozyme which specifically cleaves transcripts of endogenous genes which encode R1 proteins.
 13. Transgenic fodder plant which has an increased leaf starch content and which can be obtained by a method for generating a transgenic fodder plant with an increased leaf starch content in comparison to corresponding wild-type plants, where (a) a cell of a fodder plant is genetically modified by introducing a foreign nucleic acid molecule whose presence or expression leads to a reduced activity of an R1 protein which occurs endogenously in the plant cell; (b) a plant is regenerated from the cell generated in step (a); (c) if appropriate, further plants are generated starting from the plant generated in step (b); and (d) the foreign nucleic acid molecule according to step (a) is selected from nucleic acid molecules which, in the event of in vivo mutagenesis, lead to a mutation or an insertion of a heterologous sequence in endogenous genes which encode R1 proteins, the mutation or insertion leading to reduced expression of genes encoding R1 proteins, or to a reduced synthesis of active R1 proteins.
 14. Transgenic fodder plant which has an increased leaf starch content and which can be obtained by a method for generating a transgenic fodder plant with an increased leaf starch content in comparison to corresponding wild-type plants, where (a) a cell of a fodder plant is genetically modified by introducing a foreign nucleic acid molecule whose presence or expression leads to a reduced activity of an R1 protein which occurs endogenously in the plant cell; (b) a plant is regenerated from the cell generated in step (a); (c) if appropriate, further plants are generated starting from the plant generated in step (b); and (d) the foreign nucleic acid molecule according to step (a) is selected from DNA molecules which simultaneously encode at least one antisense RNA and at least one sense RNA, where the antisense RNA and the sense RNA form a RNA duplex which brings about a reduced expression of endogenous genes which encode R1 proteins.
 15. Transgenic plant cell which is characterized in that it originates from a transgenic fodder plant according to claim 10 and which is genetically modified with a foreign nucleic acid molecule by a method for generating a transgenic fodder plant with an increased leaf starch content in comparison to corresponding wild-type plants, where (a) a cell of a fodder plant is genetically modified by introducing a foreign nucleic acid molecule whose presence or expression leads to a reduced activity of an R1 protein which occurs endogenously in the plant cell; (b) a plant is regenerated from the cell generated in step (a); (c) if appropriate, further plants are generated starting from the plant generated in step (b); and (d) the foreign nucleic acid molecule according to step (a) is selected from DNA molecules which encode at least one antisense RNA which brings about reduced expression of endogenous genes which encode R1 proteins.
 16. Transgenic plant cell which is characterized in that it originates from a transgenic fodder plant according to claim 11 and which is genetically modified with a foreign nucleic acid molecule by a method for generating a transgenic fodder plant with an increased leaf starch content in comparison to corresponding wild-type plants, where (a) a cell of a fodder plant is genetically modified by introducing a foreign nucleic acid molecule whose presence or expression leads to a reduced activity of an R1 protein which occurs endogenously in the plant cell; (b) a plant is regenerated from the cell generated in step (a); (c) if appropriate, further plants are generated starting from the plant generated in step (b); and (d) the foreign nucleic acid molecule according to step (a) is selected from DNA molecules which, via a cosuppression effect, lead to reduced expression of endogenous genes which encode R1 proteins.
 17. Transgenic plant cell which is characterized in that it originates from a transgenic fodder plant according to claim 12 and which is genetically modified with a foreign nucleic acid molecule by a method for generating a transgenic fodder plant with an increased leaf starch content in comparison to corresponding wild-type plants, where (a) a cell of a fodder plant is genetically modified by introducing a foreign nucleic acid molecule whose presence or expression leads to a reduced activity of an R1 protein which occurs endogenously in the plant cell; (b) a plant is regenerated from the cell generated in step (a); (c) if appropriate, further plants are generated starting from the plant generated in step (b); and (d) the foreign nucleic acid molecule according to step (a) is selected from DNA molecules which encode at least one ribozyme which specifically cleaves transcripts of endogenous genes which encode R1 proteins.
 18. Transgenic plant cell which is characterized in that it originates from a transgenic fodder plant according to claim 12 and which is genetically modified with a foreign nucleic acid molecule by a method for generating a transgenic fodder plant with an increased leaf starch content in comparison to corresponding wild-type plants, where (a) a cell of a fodder plant is genetically modified by introducing a foreign nucleic acid molecule whose presence or expression leads to a reduced activity of an R1 protein which occurs endogenously in the plant cell; (b) a plant is regenerated from the cell generated in step (a); (c) if appropriate, further plants are generated starting from the plant generated in step (b); and (d) the foreign nucleic acid molecule according to step (a) is selected from nucleic acid molecules which, in the event of in vivo mutagenesis, lead to a mutation or an insertion of a heterologous sequence in endogenous genes which encode R1 proteins, the mutation or insertion leading to reduced expression of genes encoding R1 proteins, or to a reduced synthesis of active R1 proteins.
 19. Transgenic plant cell which is characterized in that it originates from a transgenic fodder plant according to claim 12 and which is genetically modified with a foreign nucleic acid molecule by a method for generating a transgenic fodder plant with an increased leaf starch content in comparison to corresponding wild-type plants, where (a) a cell of a fodder plant is genetically modified by introducing a foreign nucleic acid molecule whose presence or expression leads to a reduced activity of an R1 protein which occurs endogenously in the plant cell; (b) a plant is regenerated from the cell generated in step (a); (c) if appropriate, further plants are generated starting from the plant generated in step (b); and (d) the foreign nucleic acid molecule according to step (a) is selected from DNA molecules which simultaneously encode at least one antisense RNA and at least one sense RNA, where the antisense RNA and the sense RNA form a RNA duplex which brings about a reduced expression of endogenous genes which encode R1 proteins.
 20. Propagation material of transgenic fodder plants according to claim 10, comprising transgenic plant cells which are characterized in that they are genetically modified with a foreign nucleic acid molecule by a method for generating a transgenic fodder plant with an increased leaf starch content in comparison to corresponding wild-type plants, where (a) a cell of a fodder plant is genetically modified by introducing a foreign nucleic acid molecule whose presence or expression leads to a reduced activity of an R1 protein which occurs endogenously in the plant cell; (b) a plant is regenerated from the cell generated in step (a); (c) if appropriate, further plants are generated starting from the plant generated in step (b); and (d) the foreign nucleic acid molecule according to step (a) is selected from DNA molecules which encode at least one antisense RNA which brings about reduced expression of endogenous genes which encode R1 proteins.
 21. Propagation material of transgenic fodder plants according to claim 11, comprising transgenic plant cells which are characterized in that they are genetically modified with a foreign nucleic acid molecule by a method for generating a transgenic fodder plant with an increased leaf starch content in comparison to corresponding wild-type plants, where (a) a cell of a fodder plant is genetically modified by introducing a foreign nucleic acid molecule whose presence or expression leads to a reduced activity of an R1 protein which occurs endogenously in the plant cell; (b) a plant is regenerated from the cell generated in step (a); (c) if appropriate, further plants are generated starting from the plant generated in step (b); and (d) the foreign nucleic acid molecule according to step (a) is selected from DNA molecules which, via a cosuppression effect, lead to reduced expression of endogenous genes which encode R1 proteins.
 22. Propagation material of transgenic fodder plants according to claim 12, comprising transgenic plant cells which are characterized in that they are genetically modified with a foreign nucleic acid molecule by a method for generating a transgenic fodder plant with an increased leaf starch content in comparison to corresponding wild-type plants, where (a) a cell of a fodder plant is genetically modified by introducing a foreign nucleic acid molecule whose presence or expression leads to a reduced activity of an R1 protein which occurs endogenously in the plant cell; (b) a plant is regenerated from the cell generated in step (a); (c) if appropriate, further plants are generated starting from the plant generated in step (b); and (d) the foreign nucleic acid molecule according to step (a) is selected from DNA molecules which encode at least one ribozyme which specifically cleaves transcripts of endogenous genes which encode R1 proteins.
 23. Propagation material of transgenic fodder plants according to claim 13, comprising transgenic plant cells which are characterized in that they are genetically modified with a foreign nucleic acid molecule by a method for generating a transgenic fodder plant with an increased leaf starch content in comparison to corresponding wild-type plants, where (a) a cell of a fodder plant is genetically modified by introducing a foreign nucleic acid molecule whose presence or expression leads to a reduced activity of an R1 protein which occurs endogenously in the plant cell; (b) a plant is regenerated from the cell generated in step (a); (c) if appropriate, further plants are generated starting from the plant generated in step (b); and (d) nucleic acid molecules which, in the event of in vivo mutagenesis, lead to a mutation or an insertion of a heterologous sequence in endogenous genes which encode R1 proteins, the mutation or insertion leading to reduced expression of genes encoding R1 proteins, or to a reduced synthesis of active R1 proteins.
 24. Propagation material of transgenic fodder plants according to claim 14, comprising transgenic plant cells which are characterized in that they are genetically modified with a foreign nucleic acid molecule by a method for generating a transgenic fodder plant with an increased leaf starch content in comparison to corresponding wild-type plants, where (a) a cell of a fodder plant is genetically modified by introducing a foreign nucleic acid molecule whose presence or expression leads to a reduced activity of an R1 protein which occurs endogenously in the plant cell; (b) a plant is regenerated from the cell generated in step (a); (c) if appropriate, further plants are generated starting from the plant generated in step (b); and (d) the foreign nucleic acid molecule according to step (a) is selected from DNA molecules which simultaneously encode at least one antisense RNA and at least one sense RNA, where the antisense RNA and the sense RNA form a RNA duplex which brings about a reduced expression of endogenous genes which encode R1 proteins. 